Molecular insights into the axon guidance molecules Sidestep and Beaten path.

The transmembrane protein Sidestep (Side) functions as a substrate-bound attractant for motor axons in Drosophila. Outgrowing motor axons recognize Side via Beaten path Ia (Beat) and migrate along Side-expressing tissues. Here, we report a structure-function analysis of these guidance molecules using a variety of mutant lines and transgenic constructs. Investigation of Side mutants shows that the exchange of a single amino acid (L241H) in the second immunoglobulin domain disturbs Side function and subcellular localization. Overexpression of Side and Beat deletion constructs in S2 cells and muscles demonstrate that the first Ig domains of both proteins are necessary for their interaction. Furthermore, subcellular distributions of several Beat constructs identify functional domains and suggest a potential posttranslational processing step in ER compartments. In fact, fusing full-length Beat at both the N- and C-terminus with GFP and mCherry, respectively, shows that the N-terminal domain is transported to the plasma membrane and exposed on the cell surface, while the C-terminal domain accumulated in the nucleus. Taken together, these results give insights into the interaction of Side and Beat and imply that Beat might be subject to proteolytic cleavage during maturation.

Rearrangements in the musculature correlate with jumping behaviour in legless Mediterranean fruit fly larvae Ceratitis capitata (Tephritidae).

Larvae of holometabolic insects evolved different crawling strategies depending on the presence or absence of larval legs or life style. A rather unusual mode of locomotion has independently evolved in legless larvae of several dipteran species. Maggots of the Mediterranean fruit fly Ceratitis capitata developed an effective jumping mechanism to increase locomotion speed or to deter predators during the search for suitable pupation sites. Here, we use high-speed videography to visualize even the fastest movements during jump preparation and take-off. Quantification of kinetic and biometric parameters reveal that maggots jump up to 15-fold of their body length from a standing position and gain speed with 27 times the acceleration of gravity. Videos at high spatial resolution show the mechanism of latch formation and release in unprecedented detail. Mouth hooks insert in the caudal segment and raise a cuticular fold that serves as a handle to pressurize the body prior to launch. Since locomotion behaviour should be intrinsically linked to neuromuscular systems, we dissected third instar larvae and determined the precise pattern of abdominal muscles fibres. Compared to non-jumping dipteran larvae, such as Drosophila melanogaster, the overall arrangement is highly similar, but a few muscle fibres show characteristic re-arrangements in orientation and strength that are consistent with a role in bending and jumping. These results suggest that body wall muscles show adaptations to jumping behaviour in Ceratitis larvae, and possibly also in other species with different jumping techniques.

Dynamic monitoring of vital functions and tissue re-organization in Saturnia pavonia (Lepidoptera, Saturniidae) during final metamorphosis by non-invasive MRI.

Magnetic resonance imaging (MRI) is the key whole-body imaging technology for observing processes within a living object providing excellent resolution and contrast between soft tissues. In the present work, we exploited the non-destructive properties of MRI to track longitudinally the dynamic changes that take place in developing pupae of the Emperor Moth (Saturnia pavonia) during the last days before eclosion. While in diapause pupae, body fluid was almost homogeneously distributed over the internal compartments, as soon as wings, legs, flight muscles and the head region were fully developed, a significant redistribution of water levels occurred between thoracic and abdominal regions. During the last two days before eclosion, the developing moths transferred substantial amounts of liquid into the gut and the labial gland, and in case of females, into developing eggs. Concomitantly, the volume of the air sacs increased drastically and their expansion/compression became clearly visible in time-resolved MR images. Furthermore, besides ventilation of the tracheal system, air sacs are likely to serve as volume reservoir for liquid transfer during development of the moths inside their pupal case. In parallel, we were able to monitor noninvasively lipid consumption, cardiac activity and haemolymph circulation during final metamorphosis.

Misregulation of Drosophila Sidestep Leads to Uncontrolled Wiring of the Adult Neuromuscular System and Severe Locomotion Defects.

Holometabolic organisms undergo extensive remodelling of their neuromuscular system during metamorphosis. Relatively, little is known whether or not the embryonic guidance of molecules and axonal growth mechanisms are re-activated for the innervation of a very different set of adult muscles. Here, we show that the axonal attractant Sidestep (Side) is re-expressed during Drosophila metamorphosis and is indispensable for neuromuscular wiring. Mutations in side cause severe innervation defects in all legs. Neuromuscular junctions (NMJs) show a reduced density or are completely absent at multi-fibre muscles. Misinnervation strongly impedes, but does not completely abolish motor behaviours, including walking, flying, or grooming. Overexpression of Side in developing muscles induces similar innervation defects; for example, at indirect flight muscles, it causes flightlessness. Since muscle-specific overexpression of Side is unlikely to affect the central circuits, the resulting phenotypes seem to correlate with faulty muscle wiring. We further show that mutations in beaten path Ia (beat), a receptor for Side, results in similar weaker adult innervation and locomotion phenotypes, indicating that embryonic guidance pathways seem to be reactivated during metamorphosis.

Axon Guidance and Collective Cell Migration by Substrate-Derived Attractants.

Neurons have evolved specialized growth structures to reach and innervate their target cells. These growth cones express specific receptor molecules that sense environmental cues and transform them into steering decisions. Historically, various concepts of axon guidance have been developed to better understand how axons reach and identify their targets. The essence of these efforts seems to be that growth cones require solid substrates and that major guidance decisions are initiated by extracellular cues. These sometimes highly conserved ligands and receptors have been extensively characterized and mediate four major guidance forces: chemoattraction, chemorepulsion, contact attraction and contact repulsion. However, during development, cells, too, do migrate in order to reach molecularly-defined niches at target locations. In fact, axonal growth could be regarded as a special case of cellular migration, where only a highly polarized portion of the cell is elongating. Here, I combine several examples from genetically tractable model organisms, such as Drosophila or zebrafish, in which cells and axons are guided by attractive cues. Regardless, if these cues are secreted into the extracellular space or exposed on cellular surfaces, migrating cells and axons seem to keep close contact with these attractants and seem to detect them right at their source. Migration towards and along such substrate-derived attractants seem to be particularly robust, as genetic deletion induces obvious searching behaviors and permanent guidance errors. In addition, forced expression of these factors in ectopic tissues is highly distractive too, regardless of the pattern of other endogenous cues. Thus, guidance and migration towards and along attractive tissues is a powerful steering mechanism that exploits affinity differences to the surroundings and, in some instances, determines growth trajectories from source to target region.

Sidestep-induced neuromuscular miswiring causes severe locomotion defects in Drosophila larvae.

Mutations in motor axon guidance molecules cause aberrant projection patterns of motor nerves. As most studies in Drosophila have analysed these molecules in fixed embryos, the consequences for larval locomotion are entirely unexplored. Here, we took advantage of sidestep (side)-mutant larvae that display severe locomotion defects because of irreparable innervation errors. Mutations in side affected all motor nerve branches and all body wall regions. Innervation defects were non-stereotypical, showing unique innervation patterns in each hemisegment. Premature activation of Side in muscle precursors abrogated dorsal migration of motor nerves, resulting in larvae with a complete loss of neuromuscular junctions on dorsal-most muscles. High-speed videography showed that these larvae failed to maintain substrate contact and inappropriately raised both head and tail segments above the substrate, resulting in unique 'arching' and 'lifting' phenotypes. These results show that guidance errors in side mutants are maintained throughout larval life and are asymmetrical with respect to the bilateral body axis. Together with similar findings in mice, this study also suggests that miswiring could be an underlying cause of inherited movement disorders.

Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases.

Dominant mutations in five tRNA synthetases cause Charcot-Marie-Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNA(Gly) aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT.

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.

The PIKE homolog Centaurin gamma regulates developmental timing in Drosophila.

Phosphoinositide-3-kinase enhancer (PIKE) proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH) domain, a GTPase-activating (GAP) domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK) and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to third instar larval transition.

Redox switch for actin.

Oxidation of actin methionine residues by the oxidation-reduction enzyme Mical is known to lead to actin filament depolymerization. SelR enzymes are now shown to reduce these oxidized actin methionines, revealing a regulated redox reaction mechanism through which cells control the assembly and disassembly of actin filaments.

Cooperation of Syd-1 with Neurexin synchronizes pre- with postsynaptic assembly.

Synapse formation and maturation requires bidirectional communication across the synaptic cleft. The trans-synaptic Neurexin-Neuroligin complex can bridge this cleft, and severe synapse assembly deficits are found in Drosophila melanogaster neuroligin (Nlg1, dnlg1) and neurexin (Nrx-1, dnrx) mutants. We show that the presynaptic active zone protein Syd-1 interacts with Nrx-1 to control synapse formation at the Drosophila neuromuscular junction. Mutants in Syd-1 (RhoGAP100F, dsyd-1), Nrx-1 and Nlg1 shared active zone cytomatrix defects, which were nonadditive. Syd-1 and Nrx-1 formed a complex in vivo, and Syd-1 was important for synaptic clustering and immobilization of Nrx-1. Consequently, postsynaptic clustering of Nlg1 was affected in Syd-1 mutants, and in vivo glutamate receptor incorporation was changed in Syd-1, Nrx-1 and Nlg1 mutants. Stabilization of nascent Syd-1-Liprin-α (DLiprin-α) clusters, important to initialize active zone formation, was Nlg1 dependent. Thus, cooperation between Syd-1 and Nrx-1-Nlg1 seems to orchestrate early assembly processes between pre- and postsynaptic membranes, promoting avidity of newly forming synaptic scaffolds.

The synaptic cytoskeleton in development and disease.

The cytoskeleton forms the backbone of neuronal architecture, sustaining its form and size, subcellular compartments and cargo logistics. The synaptic cytoskeleton can be categorized in the microtubule-based core cytoskeleton and the cortical membrane skeleton. While central microtubules form the fundamental basis for the construction of elaborate neuronal processes, including axons and synapses, cortical actin filaments are generally considered to function as mediators of synapse dynamics and plasticity. More recently, the submembranous network of spectrin and ankyrin molecules has been involved in the regulation of synaptic stability and maintenance. Disruption of the synaptic cytoskeleton primarily affects the stability and maturation of synapses but also secondarily disturbs neuronal communication. Consequently, a variety of inherited diseases are accompanied by cytoskeletal malfunctions, including spastic paraplegias, spinocerebellar ataxias, and mental retardation. Since the primary reasons for many of these diseases are still unknown model organisms with a conserved repertoire of cytoskeletal elements help to understand the underlying biological mechanisms. The astonishing technical as well as genetic accessibility of synapses in Drosophila has shown that loss of the cytoskeletal architecture leads to axonal transport defects, synaptic maturation deficits, and retraction of synaptic boutons, before synaptic terminals finally detach from their target cells, suggesting that similar processes could be involved in human neuronal diseases.

Drosophila neuroligin 1 promotes growth and postsynaptic differentiation at glutamatergic neuromuscular junctions.

Precise apposition of presynaptic and postsynaptic domains is a fundamental property of all neuronal circuits. Experiments in vitro suggest that Neuroligins and Neurexins function as key regulatory proteins in this process. In a genetic screen, we recovered several mutant alleles of Drosophila neuroligin 1 (dnlg1) that cause a severe reduction in bouton numbers at neuromuscular junctions (NMJs). In accord with reduced synapse numbers, these NMJs show reduced synaptic transmission. Moreover, lack of postsynaptic DNlg1 leads to deficits in the accumulation of postsynaptic glutamate receptors, scaffold proteins, and subsynaptic membranes, while increased DNlg1 triggers ectopic postsynaptic differentiation via its cytoplasmic domain. DNlg1 forms discrete clusters adjacent to postsynaptic densities. Formation of these clusters depends on presynaptic Drosophila Neurexin (DNrx). However, DNrx binding is not an absolute requirement for DNlg1 function. Instead, other signaling components are likely involved in DNlg1 transsynaptic functions, with essential interactions organized by the DNlg1 extracellular domain but also by the cytoplasmic domain.

Searching for guidance cues: follow the Sidestep trail.

Neuronal growth cones migrate along stereotypic pathways to find and select their correct targets. Although it is widely believed that attractive and repulsive guidance molecules provide directional cues for migrating growth cones, it is still only poorly understood how these factors cooperate in a spatial and temporal manner. We have recently proposed that Drosophila motor axons recognize and follow a Sidestep-labeled substrate pathway from the ventral nerve cord to their peripheral target muscles. Sidestep (Side) is a transmembrane protein of the immunoglobulin superfamily and functions as a target-derived attractant. On motor axons, Beaten path Ia (Beat) is required to detect Side. In addition, Beat interacts with Side both genetically and biochemically, leading to the formation of heterophilic adhesion complexes in vitro. Since Side is expressed in sensory neurons, Beat-expressing motor axons fasciculate with sensory axons and use them as migratory substrates. In a similar process, motor axons contact a subset of Side-expressing glial cells, demonstrating that, during the period of axonal pathfinding, motor axons interact with all cell types that later will be the major constituents of peripheral nerves. Here, I discuss the idea that Sidestep-mediated attraction might represent the initial step for the controlled assembly of peripheral nerves.

No sidesteps on a beaten track: motor axons follow a labeled substrate pathway.

The establishment of synaptic connections between motor neurons and muscle fibers is essential for controlled body movements in any higher organism. The wiring of the neuromuscular system in Drosophila serves as a model system for the identification of key regulatory proteins that control axon guidance and target recognition. Sidestep (Side) is a transmembrane protein of the immunoglobulin superfamily and plays a pivotal role in the coordination of motor axonal guidance decisions, as it functions as a target-derived attractant. Side, however, is expressed in a highly dynamic pattern during embryogenesis, making it difficult to deduce its precise function. We have recently shown that the expression of Side strongly correlates with the actual position of motor axonal growth cones. Motor axons seem to recognize and follow Side-positive surfaces until they reach their target fields. The motor neuronal protein Beaten path Ia (Beat) is required to detect Side. In beat mutant embryos, motor axons are no longer attracted to Side-expressing tissues. In addition, Beat and Side interact biochemically, forming heterophilic adhesion complexes in vitro. Here, I discuss the model that preferential adhesion of Beat-expressing growth cones to Side-labeled substrates could be a powerful mechanism to guide motor axons.

Drosophila motor axons recognize and follow a Sidestep-labeled substrate pathway to reach their target fields.

During development of the Drosophila nervous system, migrating motor axons contact and interact with different cell types before reaching their peripheral muscle fields. The axonal attractant Sidestep (Side) is expressed in most of these intermediate targets. Here, we show that motor axons recognize and follow Side-expressing cell surfaces from the ventral nerve cord to their target region. Contact of motor axons with Side-expressing cells induces the down-regulation of Side. In the absence of Side, the interaction with intermediate targets is lost. Misexpression of Side in side mutants strongly attracts motor axons to ectopic sites. We provide evidence that, on motor axons, Beaten path Ia (Beat) functions as a receptor or part of a receptor complex for Side. In beat mutants, motor axons no longer recognize Side-expressing cell surfaces. Furthermore, Beat interacts with Side both genetically and biochemically. These results suggest that the tracing of Side-labeled cell surfaces by Beat-expressing growth cones is a major principle of motor axon guidance in Drosophila.

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.

Drosophila MICAL regulates myofilament organization and synaptic structure.

The overall size and structure of a synaptic terminal is an important determinant of its function. In a large-scale mutagenesis screen, designed to identify Drosophila mutants with abnormally structured neuromuscular junctions (NMJs), we discovered mutations in Drosophila mical, a conserved gene encoding a multi-domain protein with a N-terminal monooxygenase domain. In mical mutants, synaptic boutons do not sprout normally over the muscle surface and tend to form clusters along synaptic branches and at nerve entry sites. Consistent with high expression of MICAL in somatic muscles, immunohistochemical stainings reveal that the subcellular localization and architecture of contractile muscle filaments are dramatically disturbed in mical mutants. Instead of being integrated into a regular sarcomeric pattern, actin and myosin filaments are disorganized and accumulate beneath the plasmamembrane. Whereas contractile elements are strongly deranged, the proposed organizer of sarcomeric structure, D-Titin, is much less affected. Transgenic expression of interfering RNA molecules demonstrates that MICAL is required in muscles for the higher order arrangement of myofilaments. Ultrastructural analysis confirms that myosin-rich thick filaments enter submembranous regions and interfere with synaptic development, indicating that the disorganized myofilaments may cause the synaptic growth phenotype. As a model, we suggest that the filamentous network around synaptic boutons restrains the spreading of synaptic branches.

At the next stop sign turn right: the metalloprotease Tolloid-related 1 controls defasciculation of motor axons in Drosophila.

Navigation of motoneuronal growth cones toward the somatic musculature in Drosophila serves as a model system to unravel the molecular mechanisms of axon guidance and target selection. In a large-scale mutagenesis screen, we identified piranha, a motor axon guidance mutant that shows strong defects in the neuromuscular connectivity pattern. In piranha mutant embryos, permanent defasciculation errors occur at specific choice points in all motor pathways. Positional cloning of piranha revealed point mutations in tolloid-related 1 (tlr1), an evolutionarily conserved gene encoding a secreted metalloprotease. Ectopic expression of Tlr1 in several tissues of piranha mutants, including hemocytes, completely restores the wild-type innervation pattern, indicating that Tlr1 functions cell non-autonomously. We further show that loss-of-function mutants of related metalloproteases do not have motor axon guidance defects and that the respective proteins cannot functionally replace Tlr1. tlr1, however, interacts with sidestep, a muscle-derived attractant. Double mutant larvae of tlr1 and sidestep show an additive phenotype and lack almost all neuromuscular junctions on ventral muscles, suggesting that Tlr1 functions together with Sidestep in the defasciculation process.