The Ankyrin Repeat Domain Controls Presynaptic Localization of Drosophila Ankyrin2 and Is Essential for Synaptic Stability.

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.

Hierarchical microtubule organization controls axon caliber and transport and determines synaptic structure and stability.

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.

Ena/VASP proteins cooperate with the WAVE complex to regulate the actin cytoskeleton.

Ena/VASP proteins and the WAVE regulatory complex (WRC) regulate cell motility by virtue of their ability to independently promote actin polymerization. We demonstrate that Ena/VASP and the WRC control actin polymerization in a cooperative manner through the interaction of the Ena/VASP EVH1 domain with an extended proline rich motif in Abi. This interaction increases cell migration and enables VASP to cooperatively enhance WRC stimulation of Arp2/3 complex-mediated actin assembly in vitro in the presence of Rac. Loss of this interaction in Drosophila macrophages results in defects in lamellipodia formation, cell spreading, and redistribution of Ena to the tips of filopodia-like extensions. Rescue experiments of abi mutants also reveals a physiological requirement for the Abi:Ena interaction in photoreceptor axon targeting and oogenesis. Our data demonstrate that the activities of Ena/VASP and the WRC are intimately linked to ensure optimal control of actin polymerization during cell migration and development.

Transsynaptic coordination of synaptic growth, function, and stability by the L1-type CAM Neuroglian.

The precise control of synaptic connectivity is essential for the development and function of neuronal circuits. While there have been significant advances in our understanding how cell adhesion molecules mediate axon guidance and synapse formation, the mechanisms controlling synapse maintenance or plasticity in vivo remain largely uncharacterized. In an unbiased RNAi screen we identified the Drosophila L1-type CAM Neuroglian (Nrg) as a central coordinator of synapse growth, function, and stability. We demonstrate that the extracellular Ig-domains and the intracellular Ankyrin-interaction motif are essential for synapse development and stability. Nrg binds to Ankyrin2 in vivo and mutations reducing the binding affinities to Ankyrin2 cause an increase in Nrg mobility in motoneurons. We then demonstrate that the Nrg-Ank2 interaction controls the balance of synapse growth and stability at the neuromuscular junction. In contrast, at a central synapse, transsynaptic interactions of pre- and postsynaptic Nrg require a dynamic, temporal and spatial, regulation of the intracellular Ankyrin-binding motif to coordinate pre- and postsynaptic development. Our study at two complementary model synapses identifies the regulation of the interaction between the L1-type CAM and Ankyrin as an important novel module enabling local control of synaptic connectivity and function while maintaining general neuronal circuit architecture.

Membrane-targeted WAVE mediates photoreceptor axon targeting in the absence of the WAVE complex in Drosophila.

A tight spatial-temporal coordination of F-actin dynamics is crucial for a large variety of cellular processes that shape cells. The Abelson interactor (Abi) has a conserved role in Arp2/3-dependent actin polymerization, regulating Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous protein (WAVE). In this paper, we report that Abi exerts nonautonomous control of photoreceptor axon targeting in the Drosophila visual system through WAVE. In abi mutants, WAVE is unstable but restored by reexpression of Abi, confirming that Abi controls the integrity of the WAVE complex in vivo. Remarkably, expression of a membrane-tethered WAVE protein rescues the axonal projection defects of abi mutants in the absence of the other subunits of the WAVE complex, whereas cytoplasmic WAVE only slightly affects the abi mutant phenotype. Thus complex formation not only stabilizes WAVE, but also provides further membrane-recruiting signals, resulting in an activation of WAVE.

Abi induces ectopic sensory organ formation by stimulating EGFR signaling.

One of the central regulators coupling tyrosine phosphorylation with cytoskeletal dynamics is the Abelson interactor (Abi). Its activity regulates WASP-/WAVE mediated F-actin formation and in addition modulates the activity of the Abelson tyrosine kinase (Abl). We have recently shown that the Drosophila Abi is capable of promoting bristle development in a wasp dependent fashion. Here, we report that Drosophila Abi induces sensory organ development by modulating EGFR signaling. Expression of a membrane-tethered activated Abi protein (Abi(Myr)) leads to an increase in MAPK activity. Additionally, suppression of EGFR activity inhibits the induction of extra-sensory organs by Abi(Myr), whereas co-expression of activated Abi(Myr) and EGFR dramatically enhances the neurogenic phenotype. In agreement with this observation Abi is able to associate with the EGFR in a common complex. Furthermore, Abi binds the Abl tyrosine kinase. A block of Abl kinase-activity reduces Abi protein stability and strongly abrogates ectopic sensory organ formation induced by Abi(Myr). Concomitantly, we noted changes in tyrosine phosphorylation supporting previous reports that Abi protein stability is linked to tyrosine phosphorylation mediated by Abl.

Abi activates WASP to promote sensory organ development.

Actin polymerization is a key process for many cellular events during development. To a large extent, the formation of filamentous actin is controlled by the WASP and WAVE proteins that activate the Arp2/3 complex in different developmental processes. WAVE function is regulated through a protein complex containing Sra1, Kette and Abi. Using biochemical, cell biological and genetic tools, we show here that the Abi protein also has a central role in activating WASP-mediated processes. Abi binds WASP through its carboxy-terminal domain and acts as a potent stimulator of WASP-dependent F-actin formation. To elucidate the biological function of abi in Drosophila melanogaster, we studied bristle development, a process known to require wasp function. Reduction of abi function leads to a loss of bristles similar to that observed in wasp mutants. Activation of Abi results in the formation of ectopic bristles, a phenotype that is suppressed by a reduction of wasp activity but is not affected by the reduction of wave function. Thus, in vivo Abi may set the balance between WASP and WAVE in different actin-based developmental processes.