Coordinated mRNA translation at the synapse is increasingly recognized as a critical mechanism for neuronal regulation. Pumilio, a translational regulator, is known to be involved in neuronal homeostasis and memory formation in Drosophila. Most recently, the mammalian Pumilio homolog Pumilio-2 (Pum2) has been found to play a role in the mammalian nervous system, in particular in regulating morphology, arborization and excitability of neuronal dendrites, in vitro. However, the role of Pum2 in vivo remains unclear. Here, we report our investigation of the functional and molecular consequences of Pum2 disruption in vivo using an array of neurophysiology, behavioral and gene expression profiling techniques. We used Pum2-deficient mice to monitor in vivo brain activity using EEG and to study behavior traits, including memory, locomotor activity and nesting capacities. Because of the suspected role of Pum2 in neuronal excitability, we also examined the susceptibility to seizure induction. Finally, we used a quantitative gene expression profiling assay to identify key molecular partners of Pum2. We found that Pum2-deficient mice have abnormal behavioral strategies in spatial and object memory test. Additionally, Pum2 deficiency is associated with increased locomotor activity and decreased body weight. We also observed environmentally-induced impairment in nesting behavior. Most importantly, Pum2-deficient mice showed spontaneous EEG abnormalities and had lower seizure thresholds using a convulsing dosage of pentylenetetrazole. Finally, some genes, including neuronal ion channels, were differentially expressed in the hippocampus of Pum2-deficient mice. These findings demonstrate that Pum2 serves key functions in the adult mammalian central nervous system encompassing neuronal excitability and behavioral response to environmental challenges.
Pumilio (Pum) is a translational repressor that binds selectively to target mRNAs and recruits Nanos (Nos) as a corepressor. In the larval neuromuscular system, Pum represses expression of the translation factor eIF-4E and the glutamate receptor subunit GluRIIA. Here we show that Nos, like Pum, is expressed at the neuromuscular junction (NMJ) and in neuronal cell bodies. Surprisingly, however, Nos and Pum have divergent functions on both the pre- and postsynaptic sides of the NMJ. In nos mutant and nos RNAi larvae, the number of NMJ boutons is increased, while loss of Pum reduces bouton number. On the postsynaptic side, Nos acts in opposition to Pum in regulating the subunit composition of the glutamate receptor. NMJ active zones are associated with GluRIIA- and GluRIIB-containing receptor clusters. Loss of Nos causes downregulation of GluRIIA and increases the levels of GluRIIB. Consistent with this finding, the electrophysiological properties of NMJs lacking postsynaptic Nos suggest that they employ primarily GluRIIB-containing receptors. Nos can regulate GluRIIB in the absence of GluRIIA, suggesting that the effects of Nos on GluRIIB levels are at least partially independent of synaptic competition between GluRIIA and GluRIIB. Nos is a target for Pum repression, and Pum binds selectively to the 3' UTRs of the nos and GluRIIA mRNAs. Our results suggest a model in which regulatory interplay among Pum, Nos, GluRIIA, and GluRIIB could cause a small change in Pum activity to be amplified into a large shift in the balance between GluRIIA and GluRIIB synapses.
translational repression; glutamate receptor; neuromuscular junction; Drosophila; mRNA binding; synaptic bouton
Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodelling. Our previous work has identified the translational repressor Pumilio (Pum) as a regulator of sodium current (INa) and excitability in Drosophila motoneurons. In this current study we show that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel paralytic (para). We identify a putative binding site for Pum in the 3′ end of the para open reading frame (ORF). Characterisation of the mechanism of action of Pum, using whole-cell patch clamp and real time RT-PCR, reveals that the full length protein is required for translational repression of para mRNA. Additionally, the co-factor Nanos (Nos) is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell-type specific. Thus, Pum-dependent regulation of INa in motoneurons requires both Nos and Brat, whilst regulation in other neuronal types seemingly requires only Nos but not Brat. We also show that Pum is able to reduce the level of nos mRNA and as such identify a potential negative-feedback mechanism to protect neurons from over-activity of Pum. Finally, we show coupling between INa (para) and IK (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as a direct target of Pum represents the first ion channel to be translationally-regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3′ untranslated region of target transcripts.
Pumilio; Nanos; Brat; paralytic; aCC; RP2
Drosophila ovarian germline stem cells (GSCs) are maintained by Dpp signaling and the Pumilio (Pum) and Nanos (Nos) translational repressors. Upon division, Dpp signaling is extinguished, and Nos is downregulated in one daughter cell, causing it to switch to a differentiating cystoblast (CB). However, downstream effectors of Pum-Nos remain unknown, and how CBs lose their responsiveness to Dpp is unclear. Here, we identify Brain Tumor (Brat) as a potent differentiation factor and target of Pum-Nos regulation. Brat is excluded from GSCs by Pum-Nos but functions with Pum in CBs to translationally repress distinct targets, including the Mad and dMyc mRNAs. Regulation of both targets simultaneously lowers cellular responsiveness to Dpp signaling, forcing the cell to become refractory to the self-renewal signal. Mathematical modeling elucidates bistability of cell fate in the Brat-mediated system, revealing how autoregulation of GSC number can arise from Brat coupling extracellular Dpp regulation to intracellular interpretation.
► Pumilio and Nanos translationally repress brat mRNA in germline stem cells ► Brat promotes differentiation by limiting Dpp signaling and cellular growth ► Brat acts with Pumilio to repress translation of the Mad and dMyc mRNAs ► Modeling shows Brat creates bistability and provides robust cell-fate control
We identified Pumilio (Pum), a Drosophila translational repressor, in a computational search for metazoan proteins whose activities might be regulated by assembly into ordered aggregates. The search algorithm was based on evolutionary sequence conservation patterns observed for yeast prion proteins, which contain aggregation-prone glutamine/asparagine (Q/N)-rich domains attached to functional domains of normal amino acid composition. We examined aggregation of Pum and its nematode ortholog PUF-9 by expression in yeast. A domain of Pum containing the Q/N-rich sequence, denoted as NQ1, the entire Pum N-terminus, and the complete PUF-9 protein localize to macroscopic aggregates (foci) in yeast. NQ1 and PUF-9 can generate the yeast Pin+ trait, which is transmitted by a heritable aggregate. NQ1 also assembles into amyloid fibrils in vitro. In Drosophila, Pum regulates postsynaptic translation at neuromuscular junctions (NMJs). To assess whether NQ1 affects synaptic Pum activity in vivo, we expressed it in muscles. We found that it negatively regulates endogenous Pum, producing gene dosage-dependent pum loss-of-function NMJ phenotypes. NQ1 coexpression also suppresses lethality and NMJ phenotypes caused by overexpression of Pum in muscles. The Q/N block of NQ1 is required for these phenotypic effects. Negative regulation of Pum by NQ1 might be explained by formation of inactive aggregates, but we have been unable to demonstrate that NQ1 aggregates in Drosophila. NQ1 could also regulate Pum by a “dominant-negative” effect, in which it would block Q/N-mediated interactions of Pum with itself or with cofactors required for translational repression.
translational repression; aggregate; neuromuscular junction; amyloid; Drosophila; prion; RNA-binding protein; stress granule; synaptic bouton
In Drosophila melanogaster, specification of wing vein cells and sensory organ precursor (SOP) cells, which later give rise to a bristle, requires EGFR signaling. Here, we show that Pumilio (Pum), an RNA-binding translational repressor, negatively regulates EGFR signaling in wing vein and bristle development. We observed that loss of Pum function yielded extra wing veins and additional bristles. Conversely, overexpression of Pum eliminated wing veins and bristles. Heterozygotes for Pum produced no phenotype on their own, but greatly enhanced phenotypes caused by the enhancement of EGFR signaling. Conversely, over-expression of Pum suppressed the effects of ectopic EGFR signaling. Components of the EGFR signaling pathway are encoded by mRNAs that have Nanos Response Element (NRE)–like sequences in their 3’UTRs; NREs are known to bind Pum to confer regulation in other mRNAs. We show that these NRE-like sequences bind Pum and confer repression on a luciferase reporter in heterologous cells. Taken together, our evidence suggests that Pum functions as a negative regulator of EGFR signaling by directly targeting components of the pathway in Drosophila.
Human PUMILIO1 (PUM1) and PUMILIO2 (PUM2) are members of the PUMILIO/FBF (PUF) family that regulate specific target mRNAs posttranscriptionally. Recent studies have identified mRNA targets associated with human PUM1 and PUM2. Here we explore the structural basis of natural target RNA recognition by human PUF proteins through crystal structures of the RNA-binding domains of PUM1 and PUM2 in complex with four cognate RNA sequences including sequences from p38α and erk2 MAP kinase mRNAs. We observe three distinct modes of RNA binding around the 5th RNA base, two of which are different from the prototypical 1 repeat:1 RNA base binding mode previously identified with model RNA sequences. RNA-binding affinities of PUM1 and PUM2 are not affected dramatically by the different binding modes in vitro. However, these modes of binding create structurally variable recognition surfaces that suggest a mechanism in vivo for recruitment of downstream effector proteins defined by the PUF:RNA complex.
Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD) is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants.
The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1) RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus.
The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to date. This, along with the amino acid variability observed within their PUM-HDs, suggests that these proteins may be involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions and throughout development.
Pumilio proteins are a class of RNA-binding proteins harboring Puf domains (or PUM-HD; Pumilio-Homology Domain), named after the founding members, Pumilio (from Drosophila melanogaster) and FBF (Fem-3 mRNA-Binding Factor from Caenorhabditis elegans). The domains contain multiple tandem repeats each of which recognizes one RNA base and is comprised of 35–39 amino acids. Puf domain proteins have been reported in organisms ranging from single-celled yeast to higher multicellular eukaryotes, such as humans and plants. In yeast and animals, they are involved in a variety of posttranscriptional RNA metabolism including RNA decay, RNA transport, rRNA processing and translational repression. However, their roles in plants are largely unknown. Recently, we have characterized the first member of the Puf family of RNA-binding proteins, APUM23, in Arabidopsis. Here, we discuss and summarize the diverse roles and targets of Puf proteins previously reported in other organisms and then highlight the potential regulatory roles of Puf proteins in Arabidopsis, using our recent study as an example.
APUM23; Arabidopsis; nucleolus; Puf protein; Pumilio; rRNA processing
Transcript degradation is a widespread and important mechanism for regulating protein abundance. Two major regulators of transcript degradation are RNA Binding Proteins (RBPs) and microRNAs (miRNAs). We computationally explored whether RBPs and miRNAs cooperate to promote transcript decay. We defined five RBP motifs based on the evolutionary conservation of their recognition sites in 3′UTRs as the binding motifs for Pumilio (PUM), U1A, Fox-1, Nova, and UAUUUAU. Recognition sites for some of these RBPs tended to localize at the end of long 3′UTRs. A specific group of miRNA recognition sites were enriched within 50 nts from the RBP recognition sites for PUM and UAUUUAU. The presence of both a PUM recognition site and a recognition site for preferentially co-occurring miRNAs was associated with faster decay of the associated transcripts. For PUM and its co-occurring miRNAs, binding of the RBP to its recognition sites was predicted to release nearby miRNA recognition sites from RNA secondary structures. The mammalian miRNAs that preferentially co-occur with PUM binding sites have recognition seeds that are reverse complements to the PUM recognition motif. Their binding sites have the potential to form hairpin secondary structures with proximal PUM binding sites that would normally limit RISC accessibility, but would be more accessible to miRNAs in response to the binding of PUM. In sum, our computational analyses suggest that a specific set of RBPs and miRNAs work together to affect transcript decay, with the rescue of miRNA recognition sites via RBP binding as one possible mechanism of cooperativity.
Transcript degradation represents an important mechanism of regulation used in diverse biological processes, including during development to eliminate maternally inherited transcripts, in adult tissues to define cell lineages, and as part of signaling pathways to down-regulate unneeded transcripts. RNA binding proteins (RBPs) and microRNAs are two major classes of molecules utilized to degrade transcripts. Using computational methods, we analyzed the genomewide cooperativity between microRNA and RBP recognition sites. We observed cooperativity between Pumilio (PUM) and specific microRNAs that impacts transcript decay. Our analysis suggests that approximately seven mammalian microRNAs preferentially co-localize with PUM binding sites, and these microRNAs have recognition motifs that are reverse complements to the PUM recognition motif. Their binding sites are more likely to form RNA hairpin structures with proximal PUM recognition sites that would limit microRNA efficiency, but would be more accessible to microRNAs in response to the binding of PUM. These results indicate that rescuing microRNA recognition sites from hairpin structures may be an important role for PUM.
In the early Drosophila embryo, asymmetric distribution of transcription factors, established as a consequence of translational control of their maternally-derived mRNAs, initiates pattern formation1-4. For instance, translation of the uniformly distributed maternal hunchback (hb) mRNA is inhibited at the posterior to form an anterior-to-posterior protein concentration gradient along the longitudinal axis5, 6. Inhibition of hb mRNA translation requires an mRNP complex (the NRE-complex) that consists of Nanos (Nos), Pumilio (Pum) and Brain tumor (Brat) proteins, and the Nos responsive element (NRE) present in the 3’ UTR of hb mRNA7-9. The identity of the mRNA 5’ effector protein that is responsible for this translational inhibition remained elusive. Here we show that d4EHP, a cap-binding protein which represses caudal (cad) mRNA translation10, also inhibits hb mRNA translation by interacting simultaneously with the mRNA 5’ cap structure (m7GpppN, where N is any nucleotide)11 and Brat. Thus, by regulating Cad and Hb expression, d4EHP plays a key role in establishing anterior-posterior axis polarity in the Drosophila embryo.
The prediction of pairing between microRNAs (miRNAs) and the miRNA recognition elements (MREs) on mRNAs is expected to be an important tool for understanding gene regulation. Here, we show that mRNAs that contain Pumilio recognition elements (PRE) in the proximity of predicted miRNA-binding sites are more likely to form stable secondary structures within their 3′-UTR, and we demonstrated using a PUM1 and PUM2 double knockdown that Pumilio proteins are general regulators of miRNA accessibility. On the basis of these findings, we developed a computational method for predicting miRNA targets that accounts for the presence of PRE in the proximity of seed-match sequences within poorly accessible structures. Moreover, we implement the miRNA-MRE duplex pairing as a two-step model, which better fits the available structural data. This algorithm, called MREdictor, allows for the identification of miRNA targets in poorly accessible regions and is not restricted to a perfect seed-match; these features are not present in other computational prediction methods.
PUF family proteins are among the best-characterized regulatory RNA-binding proteins in nonmammalian species, but relatively little is known about mRNA targets or functions of mammalian PUF proteins. In this study, we used ribonomic analysis to identify and analyze mRNAs associated with ribonucleoproteins containing an endogenous human PUF protein, Pum1. Pum1-associated mRNAs were highly enriched for genes encoding proteins that function in transcriptional regulation and cell cycle/proliferation, results consistent with the posttranscriptional RNA regulon model and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of 3′ untranslated region sequences of Pum1-associated mRNAs revealed a core Pum1 consensus sequence, UGUAHAUA. Pum1 knockdown demonstrated that Pum1 enhances decay of associated mRNAs, and relocalization of Pum1 to stress granules suggested that Pum1 functions in repression of translation. This study is the first in vivo genome-wide mRNA target identification of a mammalian PUF protein and provides direct evidence that human PUF proteins regulate stability of associated mRNAs. Comparison of Pum1-associated mRNAs to mRNA targets of PUF proteins from Saccharomyces cerevisiae and Drosophila melanogaster demonstrates how a well-conserved RNA-binding domain and cognate binding sequence have been evolutionarily rewired to regulate the collective expression of different sets of functionally related genes.
Stem cells can either differentiate into more specialized cells or undergo self-renewal. Several lines of evidence from different organisms suggest that these processes depend on the post-transcriptional regulation of gene expression. The presence of the PUF [Pumilio/FBF (fem-3 binding factor)] domain defines a conserved family of RNA binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans, Pumilio-2 (PUM2) is expressed in embryonic stem cells and adult germ cells. Here we show that PUM2 is expressed in a subpopulation of adipose-derived stem cell (ASC) cultures, with a granular pattern of staining in the cytoplasm. Protein levels of PUM2 showed no changes during the differentiation of ASCs into adipocytes. Moreover, RNAi knockdown of pum2 did not alter the rate of adipogenic differentiation compared with wild-type control cells. A ribonomic approach was used to identify PUM2-associated mRNAs. Microarray analysis showed that PUM2-bound mRNAs are part of gene networks involved in cell proliferation and gene expression control. We studied pum2 expression in cell cultures with low or very high levels of proliferation and found that changes in pum2 production were dependent on the proliferation status of the cell. Transient knockdown of pum2 expression by RNAi impaired proliferation of ASCs in vitro. Our results suggest that PUM2 does not repress differentiation of ASCs but rather is involved in the positive control of ASCs division and proliferation.
Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains.
Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3′-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3′-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.
Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.
The mitogen-activated protein (MAP) kinase (MAPK) enzyme is crucial for regulation of both stem cell maintenance and tumorigenesis. Two conserved controls of MAPK include its activation by RAS signaling and a kinase cascade as well as its inactivation by MAPK phosphatases (MKPs). We identify a third mode of conserved MAPK regulation. We demonstrate that PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins repress mRNAs encoding MAPK enzymes in both the Caenorhabditis elegans germline and human embryonic stem cells. PUF proteins have emerged as conserved regulators of germline stem cells in C. elegans, Drosophila, and probably vertebrates. Their molecular mode of action relies on binding to sequence elements in the 3′ untranslated region of target mRNAs. We report that PUF proteins bind and repress mRNAs encoding C. elegans MPK-1 as well as human ERK2 and p38α. We also report that PUF repression and MKP inactivation function redundantly in the C. elegans germline to restrict MPK-1/MAPK activity and prevent germ cell apoptosis. We suggest that this dual regulation of MAPK activity by PUF and MKP proteins may be a conserved mechanism for the control of growth and differentiation during animal development and tissue homeostasis.
Information between neurons and the target cells they innervate passes through sites of functional contact called synapses. How synapses form and are altered by sensory or cognitive experience is central to understand nervous system function. Studies of synapse formation and plasticity have concentrated on a few “model” synapses. The vertebrate neuromuscular junction (NMJ), the synapse between a motoneuron in the spinal cord and a skeletal muscle fiber, is one such model synapse. The extracellular matrix proteoglycan agrin plays an essential organizing role at the NMJ. Agrin is also present at some synapses in the brain and in other organs in the periphery, but its function outside the NMJ is unclear. The core signaling pathway for agrin at the NMJ, which is still incompletely defined, includes molecules specifically involved in this cascade and molecules used in other signaling pathways in many cells. Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved components of intracellular signaling modules that control a myriad of cellular processes. This article reviews emerging evidence that suggests that MAPKs are involved in agrin signaling at the NMJ and in the putative functions of agrin in the formation of a subset of synapses in the brain.
synapse; neuromuscular junction; dendritic filopodia; ERK; CREB phosphorylation
The transcription factor AP-1 positively controls synaptic plasticity at the Drosophila neuromuscular junction. Although in motor neurons, JNK has been shown to activate AP-1, a positive regulator of growth and strength at the larval NMJ, the consequences of JNK activation are poorly studied. In addition, the downstream transcriptional targets of JNK and AP-1 signaling in the Drosophila nervous system have yet to be identified. Here, we further investigated the role of JNK signaling at this model synapse employing an activated form of JNK-kinase; and using Serial Analysis of Gene Expression and oligonucleotide microarrays, searched for candidate early targets of JNK or AP-1 dependent transcription in neurons.
Temporally-controlled JNK induction in postembryonic motor neurons triggers synaptic growth at the NMJ indicating a role in developmental plasticity rather than synaptogenesis. An unexpected observation that JNK activation also causes a reduction in transmitter release is inconsistent with JNK functioning solely through AP-1 and suggests an additional, yet-unidentified pathway for JNK signaling in motor neurons. SAGE profiling of mRNA expression helps define the neural transcriptome in Drosophila. Though many putative AP-1 and JNK target genes arose from the genomic screens, few were confirmed in subsequent validation experiments. One potentially important neuronal AP-1 target discovered, CG6044, was previously implicated in olfactory associative memory. In addition, 5 mRNAs regulated by RU486, a steroid used to trigger conditional gene expression were identified.
This study demonstrates a novel role for JNK signaling at the larval neuromuscular junction and provides a quantitative profile of gene transcription in Drosophila neurons. While identifying potential JNK/AP-1 targets it reveals the limitations of genome-wide analyses using complex tissues like the whole brain.
Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.
Analogous to synaptotagmin 1, a calcium-sensitive regulator of presynaptic vesicle fusion, synaptotagmin 4 needs both of its calcium-binding sites to regulate synaptic plasticity via postsynaptic retrograde signaling.
Ca2+ influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca2+ in triggering vesicle fusion though the Ca2+ sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca2+ influx remain unclear. In this study, we demonstrate that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca2+–binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca2+-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca2+ sensor for presynaptic vesicle fusion.
Regulation of synaptic morphology depends on endocytosis of activated growth signal receptors, but the mechanisms regulating this membrane trafficking event are unclear. Actin polymerization mediated by WASp (Wiskott-Aldrich Syndrome Protein) and the Arp2/3 (Actin related protein 2/3) complex generates forces at multiple stages of endocytosis. F-BAR/SH3 domain proteins play key roles in this process by coordinating membrane deformation with WASp-dependent actin polymerization. However, it is not known how other WASp ligands, such as the small GTPase Cdc42, coordinate with F-BAR/SH3 proteins to regulate actin polymerization at membranes. Nervous Wreck (Nwk) is a conserved neuronal F-BAR/SH3 protein that localizes to periactive zones at the Drosophila larval neuromuscular junction (NMJ) and is required for regulation of synaptic growth via BMP signaling. Here we show that Nwk interacts with the endocytic proteins dynamin and Dap160 and functions together with Cdc42 to promote WASp-mediated actin polymerization in vitro and to regulate synaptic growth in vivo. Cdc42 function is associated with Rab11-dependent recycling endosomes, and we show that Rab11 co-localizes with Nwk at the NMJ. Taken together, our results suggest that synaptic growth activated by growth factor signaling is controlled at an endosomal compartment via coordinated Nwk and Cdc42-dependent actin assembly.
actin; endocytosis; F-BAR; Drosophila; neuromuscular junction; synaptic growth
A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C5-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50–70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50–70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission.
Drosophila; synaptic transmission; synapse; presynaptic; ceramidase; ceramide; sphingolipids; lipid regulation
Growing experimental evidence suggests that mechanical tension plays a significant role in determining the growth, guidance, and function of neurons. Mechanical tension in axons contributes to neurotransmitter clustering at the Drosophila neuromuscular junction (NMJ) and is actively regulated by neurons both in vitro and in vivo. In this work, we applied mechanical strain on in vivo Drosophila neurons and in vitro Aplysia neurons and studied their vesicle dynamics by live-imaging. Our experiments show that mechanical stretch modulates the dynamics of vesicles in two different model systems: (1) The global accumulation of synaptic vesicles (SV) at the Drosophila NMJ and (2) the local motion of individual large dense core vesicles (LDCV) in Aplysia neurites. Specifically, a sustained stretch results in enhanced SV accumulation in the Drosophila NMJ. This increased SV accumulation occurs in the absence of extracellular Ca2+, plateaus after approximately 50 min, and persists for at least 30 min after stretch is reduced. On the other hand, mechanical compression in Aplysia neurites immediately disrupts LDCV motion, leading to decreased range and processivity. This impairment of LDCV motion persists for at least 15 min after tension is restored. These results show that mechanical stretch modulates both local and global vesicle dynamics and strengthens the notion that tension serves a role in regulating neuronal function.
cell mechanics; subcellular; live-imaging; vesicle tracking; Drosophila; Aplysia
Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(−) mex-3(−) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.
Caenorhabditis elegans; Translational control; RNA-binding protein; Germ cells; PUF proteins