The Ankyrin Repeat Domain Controls Presynaptic Localization of Drosophila Ankyrin2 and Is Essential for Synaptic Stability.
ABSTRACT: The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. Here, we address the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. We used a P[acman] based rescue approach to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. We show that specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and we demonstrate that these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, our data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance.
Project description: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.
Project description:The dimensions of axons and synaptic terminals determine cell-intrinsic properties of neurons; however, the cellular mechanisms selectively controlling establishment and maintenance of neuronal compartments remain poorly understood. Here, we show that two giant Drosophila Ankyrin2 isoforms, Ank2-L and Ank2-XL, and the MAP1B homolog Futsch form a membrane-associated microtubule-organizing complex that determines axonal diameter, supports axonal transport, and provides independent control of synaptic dimensions and stability. Ank2-L controls microtubule and synaptic stability upstream of Ank2-XL that selectively controls microtubule organization. Synergistically with Futsch, Ank2-XL provides three-dimensional microtubule organization and is required to establish appropriate synaptic dimensions and release properties. In axons, the Ank2-XL/Futsch complex establishes evenly spaced, grid-like microtubule organization and determines axonal diameter in the absence of neurofilaments. Reduced microtubule spacing limits anterograde transport velocities of mitochondria and synaptic vesicles. Our data identify control of microtubule architecture as a central mechanism to selectively control neuronal dimensions, functional properties, and connectivity.
Project description:In vertebrate neurons, the axon initial segment (AIS) is specialized for action potential initiation. It is organized by a giant 480 Kd variant of ankyrin G (AnkG) that serves as an anchor for ion channels and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. An unusually long exon required to encode this 480Kd variant is thought to have been inserted only recently during vertebrate evolution, so the giant ankyrin-based AIS scaffold has been viewed as a vertebrate adaptation for fast, precise signaling. We re-examined AIS evolution through phylogenomic analysis of ankyrins and by testing the role of ankyrins in proximal axon organization in a model multipolar Drosophila neuron (ddaE). We find giant isoforms of ankyrin in all major bilaterian phyla, and present evidence in favor of a single common origin for giant ankyrins and the corresponding long exon in a bilaterian ancestor. This finding raises the question of whether giant ankyrin isoforms play a conserved role in AIS organization throughout the Bilateria. We examined this possibility by looking for conserved ankyrin-dependent AIS features in Drosophila ddaE neurons via live imaging. We found that ddaE neurons have an axonal diffusion barrier proximal to the cell body that requires a giant isoform of the neuronal ankyrin Ank2. Furthermore, the potassium channel shal concentrates in the proximal axon in an Ank2-dependent manner. Our results indicate that the giant ankyrin-based cytoskeleton of the AIS may have evolved prior to the radiation of extant bilaterian lineages, much earlier than previously thought.
Project description:Synapses are intercellular junctions specialized for fast, point-to-point information transfer from a presynaptic neuron to a postsynaptic cell. At a synapse, a presynaptic terminal secretes neurotransmitters via a canonical release machinery, while a postsynaptic specialization senses neurotransmitters via diverse receptors. Synaptic junctions are likely organized by trans-synaptic cell-adhesion molecules (CAMs) that bidirectionally orchestrate synapse formation, restructuring, and elimination. Many candidate synaptic CAMs were described, but which CAMs are central actors and which are bystanders remains unclear. Moreover, multiple genes encoding synaptic CAMs were linked to neuropsychiatric disorders, but the mechanisms involved are unresolved. Here, I propose that engagement of multifarious synaptic CAMs produces parallel trans-synaptic signals that mediate the establishment, organization, and plasticity of synapses, thereby controlling information processing by neural circuits. Among others, this hypothesis implies that synapse formation can be understood in terms of inter- and intracellular signaling, and that neuropsychiatric disorders involve an impairment in such signaling.
Project description:Lethal Giant Larvae (LGL) is a cytosolic cell polarity scaffold whose loss dominantly enhances neuromuscular junction (NMJ) synaptic overgrowth caused by loss of the Fragile X Mental Retardation Protein (FMRP). However, direct roles for LGL in NMJ morphological and functional development have not before been tested. Here, we use confocal imaging and two-electrode voltage-clamp electrophysiology at the Drosophila larval NMJ to define the synaptic requirements of LGL. We find that LGL is expressed both pre- and postsynaptically, where the scaffold localizes at the membrane on both sides of the synaptic interface. We show that LGL has a cell autonomous presynaptic role facilitating NMJ terminal branching and synaptic bouton formation. Moreover, loss of both pre- and postsynaptic LGL strongly decreases evoked neurotransmission strength, whereas the frequency and amplitude of spontaneous synaptic vesicle fusion events is increased. Cell-targeted RNAi and rescue reveals separable pre- and postsynaptic LGL roles mediating neurotransmission. We show that presynaptic LGL facilitates the assembly of active zone vesicle fusion sites, and that neuronally targeted rescue of LGL is sufficient to ameliorate increased synaptic vesicle cycling imaged with FM1-43 dye labeling. Postsynaptically, we show that loss of LGL results in a net increase in total glutamate receptor (GluR) expression, associated with the selective elevation of GluRIIB subunit-containing receptors. Taken together, these data indicate that the presynaptic LGL scaffold facilitates the assembly of active zone fusion sites to regulate synaptic vesicle cycling, and that the postsynaptic LGL scaffold modulates glutamate receptor composition and function.
Project description:Drosophila Ringmaker (Ringer) is homologous to the human Tubulin Polymerization Promoting Proteins (TPPPs) that are implicated in the stabilization and bundling of microtubules (MTs) that are particularly important for neurons and are also implicated in synaptic organization and plasticity. No in vivo functional data exist that have addressed the role of TPPP in synapse organization in any system. Here, we present the phenotypic and functional characterization of ringer mutants during Drosophila larval neuromuscular junction (NMJ) synaptic development. ringer mutants show reduced synaptic growth and transmission and display phenotypic similarities and genetic interactions with the Drosophila homolog of vertebrate Microtubule Associated Protein (MAP)1B, futsch. Immunohistochemical and biochemical analyses show that individual and combined loss of Ringer and Futsch cause a significant reduction in MT loops at the NMJs and reduced acetylated-tubulin levels. Presynaptic over-expression of Ringer and Futsch causes elevated levels of acetylated-tubulin and significant increase in NMJ MT loops. These results indicate that Ringer and Futsch regulate synaptic MT organization in addition to synaptic growth. Together our findings may inform studies on the close mammalian homolog, TPPP, and provide insights into the role of MTs and associated proteins in synapse growth and organization.
Project description:The neuromuscular junction (NMJ) is a chemical synapse formed between motoneurons and skeletal muscle fibers. The vertebrate NMJ uses acetylcholine (ACh) as the neurotransmitter and features numerous invaginations of the postsynaptic muscle membrane termed junctional folds. ACh receptors (AChRs) are believed to be concentrated on the crest of junctional folds but their spatial organization remains to be fully understood. In this study, we utilized super-resolution microscopy to examine the nanoscale organization of AChRs at NMJ. Using Structured Illumination Microscopy, we found that AChRs appear as stripes within the pretzel-shaped mouse NMJs, which however, do not correlate with the size of the crests of junctional folds. By comparing the localization of AChRs with several pre- and postsynaptic markers of distinct compartments of NMJs, we found that AChRs are not distributed evenly across the crest of junctional folds as previously thought. Instead, AChR stripes are more closely aligned with the openings of junctional folds as well as with the presynaptic active zone. Using Stochastic Optical Reconstruction Microscopy (STORM) for increased resolution, we found that each AChR stripe contains an AChR-poor slit at the center that is equivalent to the size of the opening of junctional folds. Together, these findings indicate that AChRs are largely localized to the edges of crests surrounding the opening of folds to align with the presynaptic active zones. Such a nanoscale organization of AChRs potentially enables trans-synaptic alignment for effective synaptic transmission of NMJs.
Project description:Synapse remodeling is an extremely dynamic process, often regulated by neural activity. Here we show during activity-dependent synaptic growth at the Drosophila NMJ many immature synaptic boutons fail to form stable postsynaptic contacts, are selectively shed from the parent arbor, and degenerate or disappear from the neuromuscular junction (NMJ). Surprisingly, we also observe the widespread appearance of presynaptically derived "debris" during normal synaptic growth. The shedding of both immature boutons and presynaptic debris is enhanced by high-frequency stimulation of motorneurons, indicating that their formation is modulated by neural activity. Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material. Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised. Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.
Project description:Members of the Tre-2/Bub2/Cdc16 (TBC) family of proteins are believed to function as GTPase-activating proteins (GAPs) for Rab GTPases, which play pivotal roles in intracellular membrane trafficking. Although membrane trafficking is fundamental to neuronal morphogenesis and function, the roles of TBC-family Rab GAPs have been poorly characterized in the nervous system. In this paper, we provide genetic evidence that Tbc1d15-17, the Drosophila homolog of mammalian Rab7-GAP TBC1d15, is required for normal presynaptic growth and postsynaptic organization at the neuromuscular junction (NMJ). A loss-of-function mutation in Tbc1d15-17 or its presynaptic knockdown leads to an increase in synaptic bouton number and NMJ length. Tbc1d15-17 mutants are also defective in the distribution of the postsynaptic scaffold Discs-large (Dlg) and in the level of the postsynaptic glutamate subunit GluRIIA. These postsynaptic phenotypes are recapitulated by postsynaptic knockdown of Tbc1d15-17. We also show that presynaptic overexpression of a constitutively active Rab7 mutant in a wild-type background causes a synaptic overgrowth phenotype resembling that of Tbc1d15-17 mutants, while a dominant-negative form of Rab7 has the opposite effect. Together, our findings establish a novel role for Tbc1d15-17 and its potential substrate Rab7 in regulating synaptic development.
Project description:Retrograde signals induced by synaptic activities are derived from postsynaptic cells to potentiate presynaptic properties, such as cytoskeletal dynamics, gene expression, and synaptic growth. However, it is not known whether activity-dependent retrograde signals can also depotentiate synaptic properties. Here we report that laminin A (LanA) functions as a retrograde signal to suppress synapse growth at Drosophila neuromuscular junctions (NMJs). The presynaptic integrin pathway consists of the integrin subunit ?? and focal adhesion kinase 56 (Fak56), both of which are required to suppress crawling activity-dependent NMJ growth. LanA protein is localized in the synaptic cleft and only muscle-derived LanA is functional in modulating NMJ growth. The LanA level at NMJs is inversely correlated with NMJ size and regulated by larval crawling activity, synapse excitability, postsynaptic response, and anterograde Wnt/Wingless signaling, all of which modulate NMJ growth through LanA and ??. Our data indicate that synaptic activities down-regulate levels of the retrograde signal LanA to promote NMJ growth.