RESUMEN
Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution. To validate the dataset, we traced brain-spanning circuitry involving the mushroom body (MB), which has been extensively studied for its role in learning. All inputs to Kenyon cells (KCs), the intrinsic neurons of the MB, were mapped, revealing a previously unknown cell type, postsynaptic partners of KC dendrites, and unexpected clustering of olfactory projection neurons. These reconstructions show that this freely available EM volume supports mapping of brain-spanning circuits, which will significantly accelerate Drosophila neuroscience. VIDEO ABSTRACT.
Asunto(s)
Mapeo Encefálico/métodos , Conectoma/métodos , Red Nerviosa/anatomía & histología , Animales , Encéfalo/anatomía & histología , Encéfalo/diagnóstico por imagen , Dendritas , Drosophila melanogaster/anatomía & histología , Femenino , Microscopía Electrónica/métodos , Cuerpos Pedunculados , Neuronas , Olfato/fisiología , Programas InformáticosRESUMEN
The formation of synapses during neuronal development is essential for establishing neural circuits and a nervous system1. Every presynapse builds a core 'active zone' structure, where ion channels cluster and synaptic vesicles release their neurotransmitters2. Although the composition of active zones is well characterized2,3, it is unclear how active-zone proteins assemble together and recruit the machinery required for vesicle release during development. Here we find that the core active-zone scaffold proteins SYD-2 (also known as liprin-α) and ELKS-1 undergo phase separation during an early stage of synapse development, and later mature into a solid structure. We directly test the in vivo function of phase separation by using mutant SYD-2 and ELKS-1 proteins that specifically lack this activity. These mutant proteins remain enriched at synapses in Caenorhabditis elegans, but show defects in active-zone assembly and synapse function. The defects are rescued by introducing a phase-separation motif from an unrelated protein. In vitro, we reconstitute the SYD-2 and ELKS-1 liquid-phase scaffold, and find that it is competent to bind and incorporate downstream active-zone components. We find that the fluidity of SYD-2 and ELKS-1 condensates is essential for efficient mixing and incorporation of active-zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for the assembly of the synaptic active zone, before maturation into a stable final structure.
Asunto(s)
Sinapsis/química , Sinapsis/metabolismo , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Vías NerviosasRESUMEN
Homeostatic signalling systems ensure stable but flexible neural activity and animal behaviour. Presynaptic homeostatic plasticity is a conserved form of neuronal homeostatic signalling that is observed in organisms ranging from Drosophila to human. Defining the underlying molecular mechanisms of neuronal homeostatic signalling will be essential in order to establish clear connections to the causes and progression of neurological disease. During neural development, semaphorin-plexin signalling instructs axon guidance and neuronal morphogenesis. However, semaphorins and plexins are also expressed in the adult brain. Here we show that semaphorin 2b (Sema2b) is a target-derived signal that acts upon presynaptic plexin B (PlexB) receptors to mediate the retrograde, homeostatic control of presynaptic neurotransmitter release at the neuromuscular junction in Drosophila. Further, we show that Sema2b-PlexB signalling regulates presynaptic homeostatic plasticity through the cytoplasmic protein Mical and the oxoreductase-dependent control of presynaptic actin. We propose that semaphorin-plexin signalling is an essential platform for the stabilization of synaptic transmission throughout the developing and mature nervous system. These findings may be relevant to the aetiology and treatment of diverse neurological and psychiatric diseases that are characterized by altered or inappropriate neural function and behaviour.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Homeostasis , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal , Receptores de Superficie Celular/metabolismo , Semaforinas/metabolismo , Transducción de Señal , Actinas/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Femenino , Masculino , Unión Neuromuscular/metabolismo , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Receptores Presinapticos/metabolismo , Transmisión Sináptica , Vesículas Sinápticas/metabolismoRESUMEN
Associating stimuli with positive or negative reinforcement is essential for survival, but a complete wiring diagram of a higher-order circuit supporting associative memory has not been previously available. Here we reconstruct one such circuit at synaptic resolution, the Drosophila larval mushroom body. We find that most Kenyon cells integrate random combinations of inputs but that a subset receives stereotyped inputs from single projection neurons. This organization maximizes performance of a model output neuron on a stimulus discrimination task. We also report a novel canonical circuit in each mushroom body compartment with previously unidentified connections: reciprocal Kenyon cell to modulatory neuron connections, modulatory neuron to output neuron connections, and a surprisingly high number of recurrent connections between Kenyon cells. Stereotyped connections found between output neurons could enhance the selection of learned behaviours. The complete circuit map of the mushroom body should guide future functional studies of this learning and memory centre.
Asunto(s)
Encéfalo/citología , Encéfalo/fisiología , Conectoma , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Memoria/fisiología , Animales , Retroalimentación Fisiológica , Femenino , Larva/citología , Larva/fisiología , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/fisiología , Vías Nerviosas , Sinapsis/metabolismoRESUMEN
Natural events present multiple types of sensory cues, each detected by a specialized sensory modality. Combining information from several modalities is essential for the selection of appropriate actions. Key to understanding multimodal computations is determining the structural patterns of multimodal convergence and how these patterns contribute to behaviour. Modalities could converge early, late or at multiple levels in the sensory processing hierarchy. Here we show that combining mechanosensory and nociceptive cues synergistically enhances the selection of the fastest mode of escape locomotion in Drosophila larvae. In an electron microscopy volume that spans the entire insect nervous system, we reconstructed the multisensory circuit supporting the synergy, spanning multiple levels of the sensory processing hierarchy. The wiring diagram revealed a complex multilevel multimodal convergence architecture. Using behavioural and physiological studies, we identified functionally connected circuit nodes that trigger the fastest locomotor mode, and others that facilitate it, and we provide evidence that multiple levels of multimodal integration contribute to escape mode selection. We propose that the multilevel multimodal convergence architecture may be a general feature of multisensory circuits enabling complex input-output functions and selective tuning to ecologically relevant combinations of cues.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Locomoción , Vías Nerviosas/fisiología , Animales , Sistema Nervioso Central/citología , Sistema Nervioso Central/fisiología , Señales (Psicología) , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Interneuronas/metabolismo , Larva/citología , Larva/fisiología , Neuronas Motoras/metabolismo , Células Receptoras Sensoriales/metabolismo , Transducción de Señal , Sinapsis/metabolismoRESUMEN
Animal behaviour arises from computations in neuronal circuits, but our understanding of these computations has been frustrated by the lack of detailed synaptic connection maps, or connectomes. For example, despite intensive investigations over half a century, the neuronal implementation of local motion detection in the insect visual system remains elusive. Here we develop a semi-automated pipeline using electron microscopy to reconstruct a connectome, containing 379 neurons and 8,637 chemical synaptic contacts, within the Drosophila optic medulla. By matching reconstructed neurons to examples from light microscopy, we assigned neurons to cell types and assembled a connectome of the repeating module of the medulla. Within this module, we identified cell types constituting a motion detection circuit, and showed that the connections onto individual motion-sensitive neurons in this circuit were consistent with their direction selectivity. Our results identify cellular targets for future functional investigations, and demonstrate that connectomes can provide key insights into neuronal computations.
Asunto(s)
Conectoma , Drosophila/fisiología , Modelos Biológicos , Percepción de Movimiento/fisiología , Vías Visuales/fisiología , Animales , Femenino , Vías Visuales/citologíaRESUMEN
Recent findings implicate alternate core promoter recognition complexes in regulating cellular differentiation. Here we report a spatial segregation of the alternative core factor TAF3, but not canonical TFIID subunits, away from the nuclear periphery, where the key myogenic gene MyoD is preferentially localized in myoblasts. This segregation is correlated with the differential occupancy of TAF3 versus TFIID at the MyoD promoter. Loss of this segregation by modulating either the intranuclear location of the MyoD gene or TAF3 protein leads to altered TAF3 occupancy at the MyoD promoter. Intriguingly, in differentiated myotubes, the MyoD gene is repositioned to the nuclear interior, where TAF3 resides. The specific high-affinity recognition of H3K4Me3 by the TAF3 PHD (plant homeodomain) finger appears to be required for the sequestration of TAF3 to the nuclear interior. We suggest that intranuclear sequestration of core transcription components and their target genes provides an additional mechanism for promoter selectivity during differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Desarrollo de Músculos/fisiología , Regiones Promotoras Genéticas , Animales , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Proteínas de Homeodominio/metabolismo , Ratones , Desarrollo de Músculos/genética , Proteína MioD/metabolismo , Miogenina/metabolismo , Transporte de Proteínas/fisiología , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción/metabolismoRESUMEN
Focused-ion-beam scanning electron microscopy (FIB-SEM) has become an essential tool for studying neural tissue at resolutions below 10 nm × 10 nm × 10 nm, producing data sets optimized for automatic connectome tracing. We present a technical advance, ultrathick sectioning, which reliably subdivides embedded tissue samples into chunks (20 µm thick) optimally sized and mounted for efficient, parallel FIB-SEM imaging. These chunks are imaged separately and then 'volume stitched' back together, producing a final three-dimensional data set suitable for connectome tracing.
Asunto(s)
Conectoma/métodos , Imagenología Tridimensional , Microscopía Electrónica de Rastreo , Animales , Encéfalo/ultraestructuraRESUMEN
Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.
Asunto(s)
Axonema , Caenorhabditis elegans , Animales , Ratones , Axonema/metabolismo , Axonema/ultraestructura , Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Mamíferos , Microtúbulos/metabolismo , Movimiento , Tubulina (Proteína)/metabolismoRESUMEN
Brains contain networks of interconnected neurons and so knowing the network architecture is essential for understanding brain function. We therefore mapped the synaptic-resolution connectome of an entire insect brain (Drosophila larva) with rich behavior, including learning, value computation, and action selection, comprising 3016 neurons and 548,000 synapses. We characterized neuron types, hubs, feedforward and feedback pathways, as well as cross-hemisphere and brain-nerve cord interactions. We found pervasive multisensory and interhemispheric integration, highly recurrent architecture, abundant feedback from descending neurons, and multiple novel circuit motifs. The brain's most recurrent circuits comprised the input and output neurons of the learning center. Some structural features, including multilayer shortcuts and nested recurrent loops, resembled state-of-the-art deep learning architectures. The identified brain architecture provides a basis for future experimental and theoretical studies of neural circuits.
Asunto(s)
Encéfalo , Conectoma , Drosophila melanogaster , Red Nerviosa , Animales , Encéfalo/ultraestructura , Neuronas/ultraestructura , Sinapsis/ultraestructura , Drosophila melanogaster/ultraestructura , Red Nerviosa/ultraestructuraRESUMEN
Microtubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.
RESUMEN
The identification of synaptic partners is challenging in dense nerve bundles, where many processes occupy regions beneath the resolution of conventional light microscopy. To address this difficulty, we have developed GRASP, a system to label membrane contacts and synapses between two cells in living animals. Two complementary fragments of GFP are expressed on different cells, tethered to extracellular domains of transmembrane carrier proteins. When the complementary GFP fragments are fused to ubiquitous transmembrane proteins, GFP fluorescence appears uniformly along membrane contacts between the two cells. When one or both GFP fragments are fused to synaptic transmembrane proteins, GFP fluorescence is tightly localized to synapses. GRASP marks known synaptic contacts in C. elegans, correctly identifies changes in mutants with altered synaptic specificity, and can uncover new information about synaptic locations as confirmed by electron microscopy. GRASP may prove particularly useful for defining connectivity in complex nervous systems.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Sistema Nervioso/citología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Animales Modificados Genéticamente , Conducta Animal , Caenorhabditis elegans , Proteínas Portadoras/genética , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Microscopía Electrónica/métodos , Modelos Biológicos , Mutación/fisiología , Neuronas/ultraestructura , Sinapsis/ultraestructuraRESUMEN
Wnt signaling through Frizzled proteins guides posterior cells and axons in C. elegans into different spatial domains. Here we demonstrate an essential role for Wnt signaling through Ror tyrosine kinase homologs in the most prominent anterior neuropil, the nerve ring. A genetic screen uncovered cwn-2, the C. elegans homolog of Wnt5, as a regulator of nerve ring placement. In cwn-2 mutants, all neuronal structures in and around the nerve ring are shifted to an abnormal anterior position. cwn-2 is required at the time of nerve ring formation; it is expressed by cells posterior of the nerve ring, but its precise site of expression is not critical for its function. In nerve ring development, cwn-2 acts primarily through the Wnt receptor CAM-1 (Ror), together with the Frizzled protein MIG-1, with parallel roles for the Frizzled protein CFZ-2. The identification of CAM-1 as a CWN-2 receptor contrasts with CAM-1 action as a non-receptor in other C. elegans Wnt pathways. Cell-specific rescue of cam-1 and cell ablation experiments reveal a crucial role for the SIA and SIB neurons in positioning the nerve ring, linking Wnt signaling to specific cells that organize the anterior nervous system.
Asunto(s)
Axones/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crecimiento & desarrollo , Neurogénesis , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Proteínas de Caenorhabditis elegans/genética , Neuronas/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Proteínas Wnt/genéticaRESUMEN
Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila) or ribbon (vertebrates) are believed to facilitate vesicle movement to the active zone (AZ) of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc) that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D). This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp) in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.
Asunto(s)
Terminales Presinápticos/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Neuronas Motoras/enzimología , Terminales Presinápticos/ultraestructura , Transmisión SinápticaRESUMEN
Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.
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Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Microscopía de Interferencia/instrumentación , Microscopía de Interferencia/métodos , Animales , Línea Celular , Chlorocebus aethiops , Humanos , MicrotúbulosRESUMEN
Presynaptic homeostatic plasticity (PHP) adaptively regulates synaptic transmission in health and disease. Despite identification of numerous genes that are essential for PHP, we lack a dynamic framework to explain how PHP is initiated, potentiated, and limited to achieve precise control of vesicle fusion. Here, utilizing both mice and Drosophila, we demonstrate that PHP progresses through the assembly and physical expansion of presynaptic signaling foci where activated integrins biochemically converge with trans-synaptic Semaphorin2b/PlexinB signaling. Each component of the identified signaling complexes, including alpha/beta-integrin, Semaphorin2b, PlexinB, talin, and focal adhesion kinase (FAK), and their biochemical interactions, are essential for PHP. Complex integrity requires the Sema2b ligand and complex expansion includes a â¼2.5-fold expansion of active-zone associated puncta composed of the actin-binding protein talin. Finally, complex pre-expansion is sufficient to accelerate the rate and extent of PHP. A working model is proposed incorporating signal convergence with dynamic molecular assemblies that instruct PHP.
Asunto(s)
Proteínas de Drosophila , Animales , Ratones , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Terminales Presinápticos/metabolismo , Talina/metabolismo , Plasticidad Neuronal/fisiología , Drosophila/metabolismoRESUMEN
Homeostatic plasticity (HP) encompasses a suite of compensatory physiological processes that counteract neuronal perturbations, enabling brain resilience. Currently, we lack a complete description of the homeostatic processes that operate within the mammalian brain. Here, we demonstrate that acute, partial AMPAR-specific antagonism induces potentiation of presynaptic neurotransmitter release in adult hippocampus, a form of compensatory plasticity that is consistent with the expression of presynaptic homeostatic plasticity (PHP) documented at peripheral synapses. We show that this compensatory plasticity can be induced within minutes, requires postsynaptic NMDARs, and is expressed via correlated increases in dendritic spine volume, active zone area, and docked vesicle number. Further, simultaneous postsynaptic genetic reduction of GluA1, GluA2, and GluA3 in triple heterozygous knockouts induces potentiation of presynaptic release. Finally, induction of compensatory plasticity at excitatory synapses induces a parallel, NMDAR-dependent potentiation of inhibitory transmission, a cross-modal effect consistent with the anti-epileptic activity of AMPAR-specific antagonists used in humans.
Asunto(s)
Receptores de N-Metil-D-Aspartato , Sinapsis , Humanos , Animales , Sinapsis/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Hipocampo/fisiología , Homeostasis/fisiología , Neurotransmisores/metabolismo , Plasticidad Neuronal/fisiología , Mamíferos/metabolismoRESUMEN
NF-kappaB signaling has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. However, the cellular and molecular activity of NF-kappaB signaling within the nervous system remains to be clearly defined. Here, we show that the NF-kappaB and IkappaB homologs Dorsal and Cactus surround postsynaptic glutamate receptor (GluR) clusters at the Drosophila NMJ. We then show that mutations in dorsal, cactus, and IRAK/pelle kinase specifically impair GluR levels, assayed immunohistochemically and electrophysiologically, without affecting NMJ growth, the size of the postsynaptic density, or homeostatic plasticity. Additional genetic experiments support the conclusion that cactus functions in concert with, rather than in opposition to, dorsal and pelle in this process. Finally, we provide evidence that Dorsal and Cactus act posttranscriptionally, outside the nucleus, to control GluR density. Based upon our data we speculate that Dorsal, Cactus, and Pelle could function together, locally at the postsynaptic density, to specify GluR levels.
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Proteínas I-kappa B/fisiología , Quinasas Asociadas a Receptores de Interleucina-1/fisiología , FN-kappa B/fisiología , Unión Neuromuscular/metabolismo , Receptores de Glutamato/metabolismo , Alelos , Animales , Western Blotting , Citoplasma/metabolismo , Drosophila , Electrofisiología , Proteínas I-kappa B/genética , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Quinasas Asociadas a Receptores de Interleucina-1/genética , Membranas/metabolismo , Microscopía Electrónica , Músculos/inervación , Músculos/fisiología , Mutación/fisiología , FN-kappa B/genética , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Receptores de Glutamato/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinapsis/genética , Sinapsis/fisiologíaRESUMEN
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.