A retrospective analysis of phosphatase catalytic subunit gene variants in patients with rare disorders identifies novel candidate neurodevelopmental disease genes.

Rare genetic disorders represent some of the most severe and life-limiting conditions that constitute a considerable burden on global healthcare systems and societies. Most individuals affected by rare disorders remain undiagnosed, highlighting the unmet need for improved disease gene discovery and novel variant interpretation. Aberrant (de) phosphorylation can have profound pathological consequences underpinning many disease processes. Numerous phosphatases and associated proteins have been identified as disease genes, with many more likely to have gone undiscovered thus far. To begin to address these issues, we have performed a systematic survey of de novo variants amongst 189 genes encoding phosphatase catalytic subunits found in rare disease patients recruited to the 100,000 Genomes Project (100 kGP), the largest national sequencing project of its kind in the United Kingdom. We found that 49% of phosphatases were found to carry de novo mutation(s) in this cohort. Only 25% of these phosphatases have been previously linked to genetic disorders. A gene-to-patient approach matching variants to phenotypic data identified 9 novel candidate rare-disease genes: PTPRD, PTPRG, PTPRT, PTPRU, PTPRZ1, MTMR3, GAK, TPTE2, PTPN18. As the number of patients undergoing whole genome sequencing increases and information sharing improves, we anticipate that reiterative analysis of genomic and phenotypic data will continue to identify candidate phosphatase disease genes for functional validation. This is the first step towards delineating the aetiology of rare genetic disorders associated with altered phosphatase function, leading to new biological insights and improved clinical outcomes for the affected individuals and their families.

Pollution induces epigenetic effects that are stably transmitted across multiple generations.

It has been hypothesized that the effects of pollutants on phenotypes can be passed to subsequent generations through epigenetic inheritance, affecting populations long after the removal of a pollutant. But there is still little evidence that pollutants can induce persistent epigenetic effects in animals. Here, we show that low doses of commonly used pollutants induce genome-wide differences in cytosine methylation in the freshwater crustacean Daphnia pulex. Uniclonal populations were either continually exposed to pollutants or switched to clean water, and methylation was compared to control populations that did not experience pollutant exposure. Although some direct changes to methylation were only present in the continually exposed populations, others were present in both the continually exposed and switched to clean water treatments, suggesting that these modifications had persisted for 7 months (>15 generations). We also identified modifications that were only present in the populations that had switched to clean water, indicating a long-term legacy of pollutant exposure distinct from the persistent effects. Pollutant-induced differential methylation tended to occur at sites that were highly methylated in controls. Modifications that were observed in both continually and switched treatments were highly methylated in controls and showed reduced methylation in the treatments. On the other hand, modifications found just in the switched treatment tended to have lower levels of methylation in the controls and showed increase methylation in the switched treatment. In a second experiment, we confirmed that sublethal doses of the same pollutants generate effects on life histories for at least three generations following the removal of the pollutant. Our results demonstrate that even low doses of pollutants can induce transgenerational epigenetic effects that are stably transmitted over many generations. Persistent effects are likely to influence phenotypic development, which could contribute to the rapid adaptation, or extinction, of populations confronted by anthropogenic stressors.

Drosophila USP22/nonstop polarizes the actin cytoskeleton during collective border cell migration.

Polarization of the actin cytoskeleton is vital for the collective migration of cells in vivo. During invasive border cell migration in Drosophila, actin polarization is directly controlled by the Hippo signaling complex, which resides at contacts between border cells in the cluster. Here, we identify, in a genetic screen for deubiquitinating enzymes involved in border cell migration, an essential role for nonstop/USP22 in the expression of Hippo pathway components expanded and merlin. Loss of nonstop function consequently leads to a redistribution of F-actin and the polarity determinant Crumbs, loss of polarized actin protrusions, and tumbling of the border cell cluster. Nonstop is a component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex, but SAGA's histone acetyltransferase module, which does not bind to expanded or merlin, is dispensable for migration. Taken together, our results uncover novel roles for SAGA-independent nonstop/USP22 in collective cell migration, which may help guide studies in other systems where USP22 is necessary for cell motility and invasion.

A method for the permeabilization of live Drosophila melanogaster larvae to small molecules and cryoprotectants.

The larvae of Drosophila melanogaster is a model organism widely used to study the muscular and nervous systems. Drosophila larvae are surrounded by a waxy cuticle that prevents permeation by most substances. Here we develop a method to remove this layer, rendering the larvae permeable to small molecules without causing death, allowing the larvae to develop to adulthood and reproduce. Permeability was assessed using fluorescein diacetate dye uptake, and mortality upon exposure to toxic levels of ethylene glycol (EG) and Dimethyl sulfoxide (DMSO). Potential uses for this method include drug delivery, toxicity assays, cryopreservation, staining, and fixation.

SERCA directs cell migration and branching across species and germ layers.

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding.

Transcriptional responses to hyperplastic MRL signalling in Drosophila.

Recent work has implicated the actin cytoskeleton in tissue size control and tumourigenesis, but how changes in actin dynamics contribute to hyperplastic growth is still unclear. Overexpression of Pico, the only Drosophila Mig-10/RIAM/Lamellipodin adapter protein family member, has been linked to tissue overgrowth via its effect on the myocardin-related transcription factor (Mrtf), an F-actin sensor capable of activating serum response factor (SRF). Transcriptional changes induced by acute Mrtf/SRF signalling have been largely linked to actin biosynthesis and cytoskeletal regulation. However, by RNA profiling, we find that the common response to chronic mrtf and pico overexpression in wing discs was upregulation of ribosome protein and mitochondrial genes, which are conserved targets for Mrtf/SRF and are known growth drivers. Consistent with their ability to induce a common transcriptional response and activate SRF signalling in vitro, we found that both pico and mrtf stimulate expression of an SRF-responsive reporter gene in wing discs. In a functional genetic screen, we also identified deterin, which encodes Drosophila Survivin, as a putative Mrtf/SRF target that is necessary for pico-mediated tissue overgrowth by suppressing proliferation-associated cell death. Taken together, our findings raise the possibility that distinct targets of Mrtf/SRF may be transcriptionally induced depending on the duration of upstream signalling.

S100A4 Elevation Empowers Expression of Metastasis Effector Molecules in Human Breast Cancer.

Many human glandular cancers metastasize along nerve tracts, but the mechanisms involved are generally poorly understood. The calcium-binding protein S100A4 is expressed at elevated levels in human cancers, where it has been linked to increased invasion and metastasis. Here we report genetic studies in a Drosophila model to define S100A4 effector functions that mediate metastatic dissemination of mutant Ras-induced tumors in the developing nervous system. In flies overexpressing mutant RasVal12 and S100A4, there was a significant increase in activation of the stress kinase JNK and production of the matrix metalloproteinase MMP1. Genetic or chemical blockades of JNK and MMP1 suppressed metastatic dissemination associated with S100A4 elevation, defining required signaling pathway(s) for S100A4 in this setting. In clinical specimens of human breast cancer, elevated levels of the mammalian paralogs MMP2, MMP9, and MMP13 are associated with a 4- to 9-fold relative decrease in patient survival. In individual tumors, levels of MMP2 and MMP13 correlated more closely with levels of S100A4, whereas MMP9 levels correlated more closely with the S100 family member S100P. Overall, our results suggest the existence of evolutionarily conserved pathways used by S100A4 to promote metastatic dissemination, with potential prognostic and therapeutic implications for metastasis by cancers that preferentially exploit nerve tract migration routes. Cancer Res; 77(3); 780-9. ©2016 AACR.

Biophysical Analysis of the N-Terminal Domain from the Human Protein Phosphatase 1 Nuclear Targeting Subunit PNUTS Suggests an Extended Transcription Factor TFIIS-Like Fold.

Human protein phosphatase 1 nuclear targeting subunit (PNUTS) plays critical roles in DNA repair, cell growth and survival. The N-terminal domain of PNUTS mediates interactions with Tox4 and the phosphatase and tensin homolog PTEN, which are essential for the roles of this protein. To study this N-terminal domain, we have established its recombinant overproduction in E. coli utilizing NusA fusion. Upon removal of the tag, the remaining PNUTS sample is soluble and highly pure. We have characterized the domain using circular dichroism and nuclear magnetic resonance and analyzed its sequence using bioinformatics. All data agree in suggesting that the PNUTS N-terminal segment adopts a compact, globular fold rich in α-helical content, where the folded fraction is substantially larger than the previously annotated fold. We conclude that this domain adopts a single fold, likely being an extended form of the transcription factor S-II leucine/tryptophan conserved-motif. Thermal denaturation yielded a melting temperature of ~49.5 °C, confirming the stability of the fold. These findings pave the way for the molecular characterization of functional interactions mediated by the N-terminal region of PNUTS.

Targeting protein function: the expanding toolkit for conditional disruption.

A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.

Drosophila as a Potential Model for Ocular Tumors.

Drosophila has made many contributions to our understanding of cancer genes and mechanisms that have subsequently been validated in mammals. Despite anatomical differences between fly and human eyes, flies offer a tractable genetic model in which to dissect the functional importance of genetic lesions found to be affected in human ocular tumors. Here, we discuss different approaches for using Drosophila as a model for ocular cancer and how studies on ocular cancer genes in flies have begun to reveal potential strategies for therapeutic intervention. We also discuss recent developments in the use of Drosophila for drug discovery, which is coming to the fore as Drosophila models are becoming tailored to study tumor types found in the clinic.

Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo.

Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd's Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo.

PNUTS/PP1 regulates RNAPII-mediated gene expression and is necessary for developmental growth.

In multicellular organisms, tight regulation of gene expression ensures appropriate tissue and organismal growth throughout development. Reversible phosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) is critical for the regulation of gene expression states, but how phosphorylation is actively modified in a developmental context remains poorly understood. Protein phosphatase 1 (PP1) is one of several enzymes that has been reported to dephosphorylate the RNAPII CTD. However, PP1's contribution to transcriptional regulation during animal development and the mechanisms by which its activity is targeted to RNAPII have not been fully elucidated. Here we show that the Drosophila orthologue of the PP1 Nuclear Targeting Subunit (dPNUTS) is essential for organismal development and is cell autonomously required for growth of developing tissues. The function of dPNUTS in tissue development depends on its binding to PP1, which we show is targeted by dPNUTS to RNAPII at many active sites of transcription on chromosomes. Loss of dPNUTS function or specific disruption of its ability to bind PP1 results in hyperphosphorylation of the RNAPII CTD in whole animal extracts and on chromosomes. Consistent with dPNUTS being a global transcriptional regulator, we find that loss of dPNUTS function affects the expression of the majority of genes in developing 1(st) instar larvae, including those that promote proliferative growth. Together, these findings shed light on the in vivo role of the PNUTS-PP1 holoenzyme and its contribution to the control of gene expression during early Drosophila development.

Validating RNAi phenotypes in Drosophila using a synthetic RNAi-resistant transgene.

RNA interference (RNAi) is a powerful and widely used approach to investigate gene function, but a major limitation of the approach is the high incidence of non-specific phenotypes that arise due to off-target effects. We previously showed that RNAi-mediated knock-down of pico, which encodes the only member of the MRL family of adapter proteins in Drosophila, resulted in reduction in cell number and size leading to reduced tissue growth. In contrast, a recent study reported that pico knockdown leads to tissue dysmorphology, pointing to an indirect role for pico in the control of wing size. To understand the cause of this disparity we have utilised a synthetic RNAi-resistant transgene, which bears minimal sequence homology to the predicted dsRNA but encodes wild type Pico protein, to reanalyse the RNAi lines used in the two studies. We find that the RNAi lines from different sources exhibit different effects, with one set of lines uniquely resulting in a tissue dysmorphology phenotype when expressed in the developing wing. Importantly, the loss of tissue morphology fails to be complemented by co-overexpression of RNAi-resistant pico suggesting that this phenotype is the result of an off-target effect. This highlights the importance of careful validation of RNAi-induced phenotypes, and shows the potential of synthetic transgenes for their experimental validation.

Phenotype and transmission efficiency of artificial and natural male-killing Spiroplasma infections in Drosophila melanogaster.

Many insect species carry inherited Spiroplasma bacteria which act as important partners and antagonists. The nature of symbioses between Spiroplasma and insects has been most extensively studied in the interaction between male-killing Spiroplasma infection and Drosophila melanogaster. For historical reasons, these studies have largely focussed on the Spiroplasma strain known as NSRO, derived from Drosophila nebulosa and transinfected into D. melanogaster. More recently, D. melanogaster naturally infected with Spiroplasma were discovered. Whilst the well studied strain NSRO is closely related to that found natively in D. melanogaster, it is unclear whether strains from D. nebulosa reflect a natural interaction when placed in D. melanogaster. In this paper, we determine if NSRO has similar or different properties from strains of Spiroplasma naturally infecting D. melanogaster in terms of transmission efficiency and the strength and timing of male-killing. Native infections were observed to have higher transmission efficiency than introduced NSRO infections during the early phases of host reproduction, but not during late reproduction. The timing and intensity of male-killing did not differ between infection classes. As a precautionary measure, it is proposed that future work seeking to reveal the nature of coevolved Spiroplasma-Drosophila interactions use the native strain.

Drosophila pico and its mammalian ortholog lamellipodin activate serum response factor and promote cell proliferation.

MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. We report here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. We show that pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. We propose that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics.

Mars promotes dTACC dephosphorylation on mitotic spindles to ensure spindle stability.

Microtubule-associated proteins (MAPs) ensure the fidelity of chromosome segregation by controlling microtubule (MT) dynamics and mitotic spindle stability. However, many aspects of MAP function and regulation are poorly understood in a developmental context. We show that mars, which encodes a Drosophila melanogaster member of the hepatoma up-regulated protein family of MAPs, is essential for MT stabilization during early embryogenesis. As well as associating with spindle MTs in vivo, Mars binds directly to protein phosphatase 1 (PP1) and coimmunoprecipitates from embryo extracts with minispindles and Drosophila transforming acidic coiled-coil (dTACC), two MAPs that function as spindle assembly factors. Disruption of binding to PP1 or loss of mars function results in elevated levels of phosphorylated dTACC on spindles. A nonphosphorylatable form of dTACC is capable of rescuing the lethality of mars mutants. We propose that Mars mediates spatially controlled dephosphorylation of dTACC, which is critical for spindle stabilization.

Essential, overlapping and redundant roles of the Drosophila protein phosphatase 1 alpha and 1 beta genes.

Protein serine/threonine phosphatase type 1 (PP1) has been found in all eukaryotes examined to date and is involved in the regulation of many cellular functions, including glycogen metabolism, muscle contraction, and mitosis. In Drosophila, four genes code for the catalytic subunit of PP1 (PP1c), three of which belong to the PP1 alpha subtype. PP1 beta 9C (flapwing) encodes the fourth PP1c gene and has a specific and nonredundant function as a nonmuscle myosin phosphatase. PP1 alpha 87B is the major form and contributes approximately 80% of the total PP1 activity. We describe the first mutant alleles of PP1 alpha 96A and show that PP1 alpha 96A is not an essential gene, but seems to have a function in the regulation of nonmuscle myosin. We show that overexpression of the PP1 alpha isozymes does not rescue semilethal PP1 beta 9C mutants, whereas overexpression of either PP1 alpha 96A or PP1 beta 9C does rescue a lethal PP1 alpha 87B mutant combination, showing that the lethality is due to a quantitative reduction in the level of PP1c. Overexpression of PP1 beta 9C does not rescue a PP1 alpha 87B, PP1 alpha 96A double mutant, suggesting an essential PP1 alpha-specific function in Drosophila.

The nonmuscle myosin phosphatase PP1beta (flapwing) negatively regulates Jun N-terminal kinase in wing imaginal discs of Drosophila.

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1beta (PP1beta). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1alpha genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. Here, we show genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw1, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. We show that puc expression is upregulated in wing imaginal discs mutant for flw1 and pucA251 and that this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw1 enhanced by puc. Together, we show that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs.

Yeast two-hybrid screens to identify Drosophila PP1-binding proteins.

Protein phosphatase type 1 (PP1) is one of the major classes of serine/threonine protein phosphatases and has been found in all eukaryotic cells examined to date. Metazoans from Drosophila to humans have multiple genes encoding catalytic subunits of PP1 (PP1c), which are involved in a wide range of biological processes. Studies in the fruit fly Drosophila melanogaster have revealed some of the essential functions of the PP1c genes. However, the PP1c isoforms have pleiotropic and overlapping functions, making it difficult to characterize their many biological roles and identify their specific in vivo substrates. Regulatory subunits of PP1 provide the key to understanding the role of PP1, as they are responsible for directing PP1c to different intracellular locations and/or affecting their activity or substrate specificity. The existence of isoform-specific PP1 regulatory subunits might also help to explain the unique roles of different PP1 catalytic subunits. Drosophila is an excellent model organism in which to characterize the role of PP1 catalytic and regulatory subunits, because it combines molecular and biochemical approaches with powerful genetics, in a well-characterized animal model. In this chapter, we will describe how the two-hybrid system can be used to identify Drosophila PP1c-interacting proteins and study their interactions with different PP1c isoforms and variants. With the appropriate bait and library constructs, this method should also be equally applicable to identifying binding subunits of related phosphatases or PP1c from other organisms.

Towards a comprehensive analysis of the protein phosphatase 1 interactome in Drosophila.

Protein phosphatase type 1 (PP1) is one of the major classes of serine/threonine protein phosphatases, and has been found in all eukaryotic cells examined to date. Metazoans from Drosophila to humans have multiple genes encoding catalytic subunits of PP1 (PP1c), which are involved in a wide range of biological processes. Different PP1c isoforms have pleiotropic and overlapping functions; this has complicated the analysis of their biological roles and the identification of specific in vivo substrates. PP1c isoforms are associated in vivo with regulatory subunits that target them to specific locations and modify their substrate specificity and activity. The PP1c-binding proteins are therefore the key to understanding the role of PP1 in particular biological processes. The existence of isoform specific PP1c-binding subunits may also help to explain the unique roles of different PP1c isoforms. Here we report the identification of 24 genes encoding Drosophila PP1c-binding proteins in the yeast two-hybrid system. Sequence analysis identified a minimal interacting fragment and putative PP1c-binding motif for each protein, delimiting the region involved in binding to PP1c. Further two-hybrid analysis showed that virtually all of the interactors were capable of binding all Drosophila PP1c isoforms. One of the novel interactors, CG1553, was examined further and shown to interact with multiple isoforms by co-immunoprecipitation from Drosophila extracts and functional interaction with PP1c isoforms in vivo. Bioinformatic analyses implicate the putative PP1c-associated subunits in a diverse array of intracellular processes. Our identification of a large number of PP1c-binding proteins with the potential for directing PP1c's specific functions in Drosophila represents a significant step towards a full understanding of the range of PP1 complexes and function in animals.