Drosophila Neuromuscular Junction Research Articles

Presynaptic local signaling by a canonical wingless pathway regulates development of the Drosophila neuromuscular junction.

Wnt/wingless signaling contributes to the development of neuronal synapses, including the Drosophila neuromuscular junction. Loss of wg (wingless) function alters the number and structure of boutons at this model synapse. Examining Wnt/wingless signaling mechanisms, we find that a distinct pathway operates presynaptically in the motoneuron and can account for many of the effects of wingless at this synapse. This pathway includes the canonical elements arrow/LRP (low-density lipoprotein receptor-related protein), dishevelled, and the glycogen synthase kinase shaggy (GSK3) and regulates the formation of microtubule loops within synaptic boutons as well as the number of synaptic boutons. This pathway, however, appears to be independent of beta-catenin signaling and the transcriptional regulation that is most frequently downstream of these components. Instead, inhibition of shaggy is likely to act locally. This pathway thus provides a parallel mechanism to the postsynaptic activation of frizzled receptors and indicates that synaptic development results from the bidirectional influence of wingless on both presynaptic and postsynaptic structures via distinct intracellular pathways.

Activity-dependent site-specific changes of glutamate receptor composition in vivo

The subunit composition of postsynaptic non-NMDA-type glutamate receptors (GluRs) determines the function and trafficking of the receptor. Changes in GluR composition have been implicated in the homeostasis of neuronal excitability and synaptic plasticity underlying learning. Here, we imaged GluRs in vivo during the formation of new postsynaptic densities (PSDs) at Drosophila neuromuscular junctions coexpressing GluRIIA and GluRIIB subunits. GluR composition was independently regulated at directly neighboring PSDs on a submicron scale. Immature PSDs typically had large amounts of GluRIIA and small amounts of GluRIIB. During subsequent PSD maturation, however, the GluRIIA/GluRIIB composition changed and became more balanced. Reducing presynaptic glutamate release increased GluRIIA, but decreased GluRIIB incorporation. Moreover, the maturation of GluR composition correlated in a site-specific manner with the level of Bruchpilot, an active zone protein that is essential for mature glutamate release. Thus, we show that an activity-dependent, site-specific control of GluR composition can contribute to match pre- and postsynaptic assembly.

Muscle Dystroglycan Organizes the Postsynapse and Regulates Presynaptic Neurotransmitter Release at the Drosophila Neuromuscular Junction

BackgroundThe Dystrophin-glycoprotein complex (DGC) comprises dystrophin, dystroglycan, sarcoglycan, dystrobrevin and syntrophin subunits. In muscle fibers, it is thought to provide an essential mechanical link between the intracellular cytoskeleton and the extracellular matrix and to protect the sarcolemma during muscle contraction. Mutations affecting the DGC cause muscular dystrophies. Most members of the DGC are also concentrated at the neuromuscular junction (NMJ), where their deficiency is often associated with NMJ structural defects. Hence, synaptic dysfunction may also intervene in the pathology of dystrophic muscles. Dystroglycan is a central component of the DGC because it establishes a link between the extracellular matrix and Dystrophin. In this study, we focused on the synaptic role of Dystroglycan (Dg) in Drosophila.Methodology/Principal FindingsWe show that Dg was concentrated postsynaptically at the glutamatergic NMJ, where, like in vertebrates, it controls the concentration of synaptic Laminin and Dystrophin homologues. We also found that synaptic Dg controlled the amount of postsynaptic 4.1 protein Coracle and alpha-Spectrin, as well as the relative subunit composition of glutamate receptors. In addition, both Dystrophin and Coracle were required for normal Dg concentration at the synapse. In electrophysiological recordings, loss of postsynaptic Dg did not affect postsynaptic response, but, surprisingly, led to a decrease in glutamate release from the presynaptic site.Conclusion/SignificanceAltogether, our study illustrates a conservation of DGC composition and interactions between Drosophila and vertebrates at the synapse, highlights new proteins associated with this complex and suggests an unsuspected trans-synaptic function of Dg.

The hangover gene negatively regulates bouton addition at the Drosophila neuromuscular junction

The synaptic growth of neurons during the development and adult life of an animal is a very dynamic and highly regulated process. During larval development in Drosophila new boutons and branches are added at the glutamatergic neuromuscular junction (NMJ) until a balance between neuronal activity and morphological structures is reached. Analysis of several Drosophila mutants suggest that bouton number and size might be regulated by separate signaling processes [Budnik, V., 1996. Synapse maturation and structural plasticity at Drosophila neuromuscular junctions. Curr. Opin. Neurobiol. 6, 858–867.]. Here we show a new role for Hangover as a negative regulator of bouton number at the NMJ. The hangover gene ( hang) encodes a nuclear zinc finger protein. It has a function in neuronal plasticity mediating ethanol tolerance, a behavior that develops upon previous experience with ethanol. hang AE10 mutants have more boutons and an extended synaptic span. Moreover, Hang expression in the motoneuron is required for the regulation of bouton number and the overall length of muscle innervation. However, the increase in bouton number does not correlate with a change in synaptic transmission, suggesting a mechanism independent from neuronal activity leads to the surplus of synaptic boutons. In contrast, we find that expression levels of the cell adhesion molecule Fasciclin II (FASII) are reduced in the hang mutant. This finding suggests that the increase in bouton number in hang mutants is caused by a reduction in FASII expression, thus, linking the regulation of nuclear gene expression with the addition of boutons at the NMJ regulated by cell adhesion molecules.

A PI3K-mediated negative feedback regulates Drosophila motor neuron excitability

AbstractNegative feedback can act as a homeostatic mechanism to maintain neuronal activity at a particular specified value. At the Drosophila neuromuscular junction, a mutation in the type II metabotropic glutamate receptor gene (mGluRA) increased motor neuron excitability by disrupting an autocrine, glutamate-mediated negative feedback. We show that mGluRA mutations increase neuronal excitability by preventing PI3 kinase (PI3K) activation and consequently hyperactivating the transcription factor Foxo. Furthermore, glutamate application increases levels of phospho-Akt, a product of PI3K signaling, within motor nerve terminals in an mGluRA-dependent manner. In humans, PI3K and type II mGluRs are implicated in epilepsy, neurofibromatosis, autism, schizophrenia and other neurological disorders; however, neither the link between type II mGluRs and PI3K, nor the role of Foxo in the control of neuronal excitability, had been previously reported. Our work suggests that some of the deficits in these neurological disorders might result from disruption of glutamate-mediated homeostasis of neuronal excitability.

Drosophila Ankyrin 2 Is Required for Synaptic Stability

Synaptic connections are stabilized through transsynaptic adhesion complexes that are anchored in the underlying cytoskeleton. The Drosophila neuromuscular junction (NMJs) serves as a model system to unravel genes required for the structural remodeling of synapses. In a mutagenesis screen for regulators of synaptic stability, we recovered mutations in Drosophila ankyrin 2 (ank2) affecting two giant Ank2 isoforms that are specifically expressed in the nervous system and associate with the presynaptic membrane cytoskeleton. ank2 mutant larvae show severe deficits in the stability of NMJs, resulting in a reduction in overall terminal size, withdrawal of synaptic boutons, and disassembly of presynaptic active zones. In addition, lack of Ank2 leads to disintegration of the synaptic microtubule cytoskeleton. Microtubules and microtubule-associated proteins fail to extend into distant boutons. Interestingly, Ank2 functions downstream of spectrin in the anchorage of synaptic microtubules, providing the cytoskeletal scaffold that is essential for synaptic stability.

A Presynaptic Giant Ankyrin Stabilizes the NMJ through Regulation of Presynaptic Microtubules and Transsynaptic Cell Adhesion

In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.

Clathrin Dependence of Synaptic-Vesicle Formation at the Drosophila Neuromuscular Junction

Among the most prominent molecular constituents of a recycling synaptic vesicle is the clathrin triskelion, composed of clathrin light chain (Clc) and clathrin heavy chain (Chc). Remarkably, it remains unknown whether clathrin is strictly necessary for the stimulus-dependent re-formation of a synaptic vesicle and, conversely, whether clathrin-independent vesicle endocytosis exists at the neuronal synapse. We employ FlAsH-FALI-mediated protein photoinactivation to rapidly (3 min) and specifically disrupt Clc function at the Drosophila neuromuscular junction. We first demonstrate that Clc photoinactivation does not impair synaptic-vesicle fusion. We then provide electrophysiological and ultrastructural evidence that synaptic vesicles, once fused with the plasma membrane, cannot be re-formed after Clc photoinactivation. Finally, we demonstrate that stimulus-dependent membrane internalization occurs after Clc photoinactivation. However, newly internalized membrane fails to resolve into synaptic vesicles. Rather, newly internalized membrane forms large and extensive internal-membrane compartments that are never observed at a wild-type synapse. We make three major conclusions. (1) FlAsH-FALI-mediated protein photoinactivation rapidly and specifically disrupts Clc function with no effect on synaptic-vesicle fusion. (2) Synaptic-vesicle re-formation does not occur after Clc photoinactivation. By extension, clathrin-independent "kiss-and-run" endocytosis does not sustain synaptic transmission during a stimulus train at this synapse. (3) Stimulus-dependent, clathrin-independent membrane internalization exists at this synapse, but it is unable to generate fusion-competent, small-diameter synaptic vesicles.

Isoflurane Reduces Excitability of Drosophila Larval Motoneurons by Activating a Hyperpolarizing Leak Conductance

Mechanisms of anesthetic-mediated presynaptic inhibition are incompletely understood. Isoflurane reduces presynaptic excitability at the larval Drosophila neuromuscular junction, slowing conduction velocity and depressing glutamate release. Mutations in the Para voltage-gated Na channel enhance anesthetic sensitivity of adult flies. Here, the author examines the role of para in anesthetic sensitivity and seeks to identify the conductance underlying presynaptic inhibition at this synapse. Neuromuscular transmission was studied using a two-electrode voltage clamp, with isoflurane applied in physiologic saline. The relation between ionic conductances and presynaptic function was modeled in the Neuron Simulation Environment. Motoneuron ionic currents were monitored via whole cell recordings. Presynaptic inhibition by isoflurane was enhanced significantly in para mutants. Computer simulations of presynaptic actions of anesthetics indicated that each candidate target conductance would have diagnostic effects on the relation between latency and amplitude of synaptic currents. The experimental latency-amplitude relation for isoflurane most closely resembled activation of a simulated hyperpolarizing leak. Simulations indicated that increased isoflurane potency in para axons resulted from reduced excitability of mutant axons. In whole cell recordings, isoflurane activated a hyperpolarizing leak current. The effects of isoflurane at the neuromuscular junction were insensitive to low pH. The effects of isoflurane on presynaptic excitability are mediated via an acid-insensitive inhibitory leak conductance. para mutations enhance the sensitivity of this anesthetic-modulated neural pathway by reducing axonal excitability. This work provides a link between anesthetic-sensitive leak currents and presynaptic function and has generated new tools for analysis of the function of this synapse.

Visualizing glutamatergic cell bodies and synapses in Drosophila larval and adult CNS

Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system (CNS) and at Drosophila neuromuscular junctions (NMJs). Although glutamate is also used as a transmitter in the Drosophila CNS, there has been no systematic description of the central glutamatergic signaling system in the fly. With the recent cloning of the Drosophila vesicular glutamate transporter (DVGLUT), it is now possible to mark many, if not all, central glutamatergic neurons and synapses. Here we present the pattern of glutamatergic synapses and cell bodies in the late larval CNS and in the adult fly brain by using an anti-DVGLUT antibody. We also introduce two new tools for studying the Drosophila glutamatergic system: a dvglut promoter fragment fused to Gal4 whose expression labels glutamatergic neurons and a green fluorescent protein (GFP)-tagged DVGLUT transgene that localizes to synapses. In the larval CNS, we find synaptic DVGLUT immunoreactivity prominent in all brain lobe neuropil compartments except for the mushroom body. Likewise in the adult CNS, glutamatergic synapses are abundant throughout all major brain structures except the mushroom body. We also find that the larval ventral nerve cord neuropil is rich in glutamatergic synapses, which are primarily located near the dorsal surface of the neuropil, segregated from the ventrally positioned cholinergic processes. This description of the glutamatergic system in Drosophila highlights the prevalence of glutamatergic neurons in the CNS and presents tools for future study and manipulation of glutamatergic transmission.

Signaling for Vesicle Mobilization and Synaptic Plasticity

The hypothesis that release of classical neurotransmitters and neuropeptides is facilitated by increasing the mobility of small synaptic vesicles (SSVs) and dense core vesicles (DCVs) could not be tested until the advent of methods for visualizing these secretory vesicles in living nerve terminals. In fact, fluorescence imaging studies have only since 2005 established that activity increases secretory vesicle mobility in motoneuron terminals and chromaffin cells. Mobilization of DCVs and SSVs appears to be due to liberation of hindered vesicles to promote quicker diffusion. However, F-actin and synapsin, which have been featured in mobilization models, are not required for activity-dependent increases in the mobility of DCVs or SSVs. Most recently, the signaling required for sustained mobilization has been identified for Drosophila motoneuron DCVs and shown to increase synaptic transmission. Specifically, presynaptic endoplasmic reticulum ryanodine receptor-mediated Ca2+ release activates Ca2+/calmodulin-dependent kinase II to mobilize DCVs and induce post-tetanic potentiation (PTP) of neuropeptide release in the Drosophila neuromuscular junction. The shared signaling for increasing vesicle mobility and PTP links vesicle mobilization and synaptic plasticity.

Effects of Hyperkinetic, a β Subunit of Shaker Voltage-Dependent K+ Channels, on the Oxidation State of Presynaptic Nerve Terminals

The Drosophila Hyperkinetic (Hk) gene encodes a β subunit of Shaker (Sh) K+ channels and shows high sequence homology to aldoketoreductase. Hk mutations are known to modify the voltage dependence and kinetics of Sh currents, which are also influenced by the oxidative state of the N-terminus region of the Sh channel, as demonstrated in heterologous expression experiments in frog oocytes. However, an in vivo role of Hk in cellular reduction/oxidation (redox) has not been demonstrated. By using a fluorescent indicator of reactive oxygen species (ROS), dihydrorhodamine-123 (DHR), we show that the presynaptic nerve terminal of larval motor axons is metabolically active, with more rapid accumulation of ROS in comparison with muscle cells. In Hk terminals, DHR fluorescence was greatly enhanced, indicating increased ROS levels. This observation implicates a role of the Hk β subunit in redox regulation in presynaptic terminals. This phenomenon was paralleled by the expected effects of the mutations affecting glutathione S-transferase S1 as well as applying H2O2 to wild-type synaptic terminals. Thus, our results also establish DHR as a useful tool for detecting ROS levels in the Drosophila neuromuscular junction.

Mathematical modeling of the Drosophila neuromuscular junction

Event Abstract Back to Event Mathematical modeling of the Drosophila neuromuscular junction Markus Knodel1*, Daniel Bucher2, Christoph Schuster2 and Gabriel Wittum1 1 Frankfurt University , Germany 2 BGCN Heidelberg, Germany An important challenge in neuroscience today is understanding how networks of neurons go about processing information. Synapses are known to be important for this process however quantitative and mathematical models of the underlying physiologic processes which occur at synaptic active zones is lacking. To counteract this problem, we are developing mathematical models of synaptic vesicle dynamics at a well characterized model synapse, the Drosophila larval neuromuscular junction. This synapses simplicity, accessibility to various electrophysiological recording and imaging techniques, and the genetic malleability which is intrinsic to the Drosophila system make it ideal for computational and mathematical studies. We have employed a reductionist approach and started by modeling single presynaptic boutons. Synaptic vesicles can be divided into different "pools" however a quantitative understanding of their dynamics at the Drosophila neuromuscular junction is lacking. We performed biologically realistic simulations of two bouton types characterized by partial differential equations - PDEs - taking into account not only the evolution in time but moreover also the spatial structure of two dimensions (the extension to the full three dimensions will be implemented soon). These PDEs are solved using UG. UG is a program library for the calculation of multi-dimensional PDEs which are solved using a finite volume approach and implicit time stepping methods leading to extended linear equation systems which can be solved by using multi grid methods. Numerical calculations are done on multi-processor computers allowing for fast calculations using different parameters in order to asses the biological feasibility of different models. In preliminary simulations, we modeled vesicle dynamics as a diffusion process describing exocytosis as Neumann streams at synaptic active zones. The results obtained with these models are consistent with experimental results however this should be regarded as a work in progress. Further refinements will be implemented such as models of glutamate diffusion and clearance from the synaptic cleft, post synaptic receptor kinetics, and physiologically realistic whole neuromuscular junction simulations. Conference: Bernstein Symposium 2008, Munich, Germany, 8 Oct - 10 Oct, 2008. Presentation Type: Poster Presentation Topic: All Abstracts Citation: Knodel M, Bucher D, Schuster C and Wittum G (2008). Mathematical modeling of the Drosophila neuromuscular junction. Front. Comput. Neurosci. Conference Abstract: Bernstein Symposium 2008. doi: 10.3389/conf.neuro.10.2008.01.074 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Nov 2008; Published Online: 17 Nov 2008. * Correspondence: Markus Knodel, Frankfurt University, Frankfurt, Germany, Markus.Knodel@iwr.uni-heidelberg.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Markus Knodel Daniel Bucher Christoph Schuster Gabriel Wittum Google Markus Knodel Daniel Bucher Christoph Schuster Gabriel Wittum Google Scholar Markus Knodel Daniel Bucher Christoph Schuster Gabriel Wittum PubMed Markus Knodel Daniel Bucher Christoph Schuster Gabriel Wittum Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Identification and investigation of Drosophila postsynaptic density homologs.

AMPA receptors are responsible for fast excitatory transmission in the CNS and the trafficking of these receptors has been implicated in LTP and learning and memory. These receptors reside in the postsynaptic density, a network of proteins that links the receptors to downstream signaling components and to the neuronal cytoskeleton. To determine whether the fruit fly, Drosophila melanogaster, possesses a similar array of proteins as are found at the mammalian PSD, we identified Drosophila homologs of 95.8% of mammalian PSD proteins. We investigated, for the first time, the role of one of these PSD proteins, Pod1 in GluR cluster formation at the Drosophila neuromuscular junction and found that mutations in pod1 resulted in a specific loss of A-type receptors at the synapse.

FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.

To maintain transmitter release during intense stimulation, neurons need to efficiently recycle vesicles at the synapse. Following membrane fusion, vesicles are reshaped and formed from the plasma membrane by bulk or clathrin-mediated endocytosis. Most synapses, including the Drosophila neuromuscular junction (NMJ), can also recycle synaptic vesicles directly by closing the fusion pore, a process referred to as "kiss and run." While the process of clathrin-mediated vesicle retrieval is under intense investigation, the kiss-and-run phenomenon remains much less accepted. To gain better insight into the mechanisms of synaptic vesicle recycling, it is therefore critical not only to identify and characterize novel players involved in the process, but also to develop novel methods to study vesicle recycling. Although in recent years numerous techniques to study vesicle traffic have been developed (see also this volume), in this chapter we outline established procedures that use the fluorescent dye FM 1-43 or related compounds to study vesicle cycling. We describe how FM 1-43 can be used to study and visualize clathrin-mediated or bulk endocytosis from the presynaptic membrane as well as exocytosis of labeled vesicles at the Drosophila NMJ, one of the best-characterized model synapses to study synaptic function in a genetic model system.

Innervation and activity dependent dynamics of postsynaptic oxidative metabolism

Despite extensive investigations into the mechanisms of aerobic respiration in mitochondria, the spontaneous metabolic activity of individual cells within a whole animal has not been observed in real time. Consequently, little is known about whether and how the level of mitochondrial energy metabolism is regulated in a cell during development of intact systems. Here we studied the dynamics of postsynaptic oxidative metabolism by monitoring the redox state of mitochondrial flavoproteins, an established indicator of energy metabolism, at the developing Drosophila neuromuscular junction. We detected transient and spatially synchronized flavoprotein autofluorescence signals in postsynaptic muscle cells. These signals were dependent on the energy substrates and coupled to changes in mitochondrial membrane potential and Ca 2+ concentration. Notably, the rate of autofluorescence signals increased during synapse formation through contact with the motoneuronal axon. This rate was also influenced by the magnitude of synaptic inputs. Thus, presynaptic cells tightly regulate postsynaptic energy metabolism presumably to maintain an energetic balance during neuromuscular synaptogenesis. Our results suggest that flavoprotein autofluorescence imaging should allow us to begin assessing the progress of synapse formation from a metabolic perspective.

Derailed regulates development of the Drosophila neuromuscular junction.

Neural function is dependent upon the proper formation and development of synapses. We show here that Wnt5 regulates the growth of the Drosophila neuromuscular junction (NMJ) by signaling through the Derailed receptor. Mutations in both wnt5 and drl result in a significant reduction in the number of synaptic boutons. Cell-type specific rescue experiments show that wnt5 functions in the presynaptic motor neuron while drl likely functions in the postsynaptic muscle cell. Epistatic analyses indicate that drl acts downstream of wnt5 to promote synaptic growth. Structure-function analyses of the Drl protein indicate that normal synaptic growth requires the extracellular Wnt inhibitory factor domain and the intracellular domain, which includes an atypical kinase. Our findings reveal a novel signaling mechanism that regulates morphology of the Drosophila NMJ.

Wnt4 Is a Local Repulsive Cue that Determines Synaptic Target Specificity

How synaptic specificity is molecularly coded in target cells is a long-standing question in neuroscience. Whereas essential roles of several target-derived attractive cues have been shown, less is known about the role of repulsion by nontarget cells. We conducted single-cell microarray analysis of two neighboring muscles (M12 and M13) in Drosophila, which are innervated by distinct motor neurons, by directly isolating them from dissected embryos. We identified a number of potential target cues that are differentially expressed between the two muscles, including M13-enriched Wnt4. When the functions of Wnt4, or putative receptors Frizzled 2 and Derailed-2 or Dishevelled were inhibited, motor neurons that normally innervate M12 (MN12s) formed smaller synapses on M12 but instead formed ectopic nerve endings on M13. Conversely, ectopic expression of Wnt4 in M12 inhibits synapse formation by MN12s. These results suggest that Wnt4, via Frizzled 2, Derailed-2, and Dishevelled, generates target specificity by preventing synapse formation on a nontarget muscle. Ectopic expression of five other M13-enriched genes, including beat-IIIc and Glutactin, also inhibits synapse formation by MN12s. These results demonstrate an important role for local repulsion in regulating cell-to-cell target specificity.

Neuronal Morphogenesis Is Regulated by the Interplay between Cyclin-Dependent Kinase 5 and the Ubiquitin Ligase Mind Bomb 1

Neuronal communication requires the coordinated assembly of polarized structures including axons, dendrites, and synapses. Here, we report the identification of a ubiquitin ligase mind bomb 1 (Mib1) in the postsynaptic density and the characterization of its role in neuronal morphogenesis. Expression of Mib1 inhibits neurite outgrowth in cell culture and its gene deletion enhances synaptic growth at the neuromuscular junction in Drosophila. The analysis of Mib1 interactome by mass spectrometry revealed that Mib1 primarily interacts with membrane trafficking proteins [e.g., EEA1 (early endosomal antigen 1), Rab11-interacting proteins, and SNAP25 (synaptosomal-associated protein of 25 kDa)-like protein] and cell adhesion components (e.g., catenin, coronin, dystrobrevin, and syndecan), consistent with its previously reported function in protein sorting. More interestingly, Mib1 is associated with deubiquitinating enzymes, BRCC36 and the mammalian ortholog of fat facets, and a number of kinases, such as casein kinase II, MARK (microtubule affinity regulating kinase)/PAR1, and cyclin-dependent kinase 5 (CDK5). Further characterization of the Mib1-CDK5 interaction indicated that the N-terminal domain of Mib1 directly binds to the regulatory subunit p35 of the CDK5 complex. In cell culture, Mib1 induces the relocalization of p35/CDK5 without affecting its degradation. Surprisingly, p35/CDK5 downregulates the protein level of Mib1 by its kinase activity, and completely rescues the Mib1-induced inhibitory effect on neurite morphology. p35/CDK5 also genetically interacts with Mib1 in the fly according to the rough-eye phenotype. The data strongly support that the negative interplay between Mib1 and p35/CDK5 may integrate the activities of multiple pathways during neuronal development.

DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth

BackgroundThe growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, we have identified DFsn as a binding partner of Highwire and characterized the roles of DFsn in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance.ResultsWe identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants – there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth.ConclusionThe F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. We propose that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses.