Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (10)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Nature Neuroscience
- Neuron
- Science (New York, N.Y.)
- The Journal of Cell Biology
- Nature Protocols
- Nature Neuroscience
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- The EMBO Journal
- Molecular Microbiology
Articles by Tobias M. Rasse in JoVE
JoVE General
In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution
1Junior Research Group Synaptic Plasticity, Hertie Institute for Clinical Brain Research, University of Tübingen, 2Graduate School of Cellular and Molecular Neuroscience, University of Tübingen
This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.
Other articles by Tobias M. Rasse on PubMed
Four Different Subunits Are Essential for Expressing the Synaptic Glutamate Receptor at Neuromuscular Junctions of Drosophila
Three ionotropic glutamate receptor subunits, designated GluRIIA, GluRIIB, and GluRIII, have been identified at neuromuscular junctions of Drosophila. Whereas GluRIIA and GluRIIB are redundant for viability, it was shown recently that GluRIII is essential for both the synaptic localization of GluRIIA and GluRIIB and the viability of Drosophila. Here we identify a fourth and a fifth subunit expressed in the neuromuscular system, which we name GluRIID and GluRIIE. Both new subunits we show to be necessary for survival. Moreover, both GluRIID and GluRIIE are required for the synaptic expression of all other glutamate receptor subunits. All five subunits are interdependent for receptor function, synaptic receptor expression, and viability. This indicates that synaptic glutamate receptors incorporate the GluRIII, GluRIID, and GluRIIE subunit together with either GluRIIA or GluRIIB at the Drosophila neuromuscular junction. At this widely used model synapse, the assembly of four different subunits to form an individual glutamate receptor channel may thus be obligatory. This study opens the way for a further characterization of in vivo glutamate receptor assembly and trafficking using the efficient genetics of Drosophila.
Glutamate Receptor Dynamics Organizing Synapse Formation in Vivo
Insight into how glutamatergic synapses form in vivo is important for understanding developmental and experience-triggered changes of excitatory circuits. Here, we imaged postsynaptic densities (PSDs) expressing a functional, GFP-tagged glutamate receptor subunit (GluR-IIA(GFP)) at neuromuscular junctions of Drosophila melanogaster larvae for several days in vivo. New PSDs, associated with functional and structural presynaptic markers, formed independently of existing synapses and grew continuously until reaching a stable size within hours. Both in vivo photoactivation and photobleaching experiments showed that extrasynaptic receptors derived from diffuse, cell-wide pools preferentially entered growing PSDs. After entering PSDs, receptors were largely immobilized. In comparison, other postsynaptic proteins tested (PSD-95, NCAM and PAK homologs) exchanged faster and with no apparent preference for growing synapses. We show here that new glutamatergic synapses form de novo and not by partitioning processes from existing synapses, suggesting that the site-specific entry of particular glutamate receptor complexes directly controls the assembly of individual PSDs.
Bruchpilot, a Protein with Homology to ELKS/CAST, is Required for Structural Integrity and Function of Synaptic Active Zones in Drosophila
Neurotransmitters are released at presynaptic active zones (AZs). In the fly Drosophila, monoclonal antibody (MAB) nc82 specifically labels AZs. We employ nc82 to identify Bruchpilot protein (BRP) as a previously unknown AZ component. BRP shows homology to human AZ protein ELKS/CAST/ERC, which binds RIM1 in a complex with Bassoon and Munc13-1. The C terminus of BRP displays structural similarities to multifunctional cytoskeletal proteins. During development, transcription of the bruchpilot locus (brp) coincides with neuronal differentiation. Panneural reduction of BRP expression by RNAi constructs permits a first functional characterization of this large AZ protein: larvae show reduced evoked but normal spontaneous transmission at neuromuscular junctions. In adults, we observe loss of T bars at active zones, absence of synaptic components in electroretinogram, locomotor inactivity, and unstable flight (hence "bruchpilot"-crash pilot). We propose that BRP is critical for intact AZ structure and normal-evoked neurotransmitter release at chemical synapses of Drosophila.
Bruchpilot Promotes Active Zone Assembly, Ca2+ Channel Clustering, and Vesicle Release
The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) were entirely lost, Ca2+ channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity.
The Ig Cell Adhesion Molecule Basigin Controls Compartmentalization and Vesicle Release at Drosophila Melanogaster Synapses
Synapses can undergo rapid changes in size as well as in their vesicle release function during both plasticity processes and development. This fundamental property of neuronal cells requires the coordinated rearrangement of synaptic membranes and their associated cytoskeleton, yet remarkably little is known of how this coupling is achieved. In a GFP exon-trap screen, we identified Drosophila melanogaster Basigin (Bsg) as an immunoglobulin domain-containing transmembrane protein accumulating at periactive zones of neuromuscular junctions. Bsg is required pre- and postsynaptically to restrict synaptic bouton size, its juxtamembrane cytoplasmic residues being important for that function. Bsg controls different aspects of synaptic structure, including distribution of synaptic vesicles and organization of the presynaptic cortical actin cytoskeleton. Strikingly, bsg function is also required specifically within the presynaptic terminal to inhibit nonsynchronized evoked vesicle release. We thus propose that Bsg is part of a transsynaptic complex regulating synaptic compartmentalization and strength, and coordinating plasma membrane and cortical organization.
Live Imaging of Synapse Development and Measuring Protein Dynamics Using Two-color Fluorescence Recovery After Photo-bleaching at Drosophila Synapses
Here we describe how to anesthetize and image Drosophila larvae as to follow 'the life history' of identified synapses and synaptic components. This protocol is sensitive, for example, the distribution of glutamate receptors expressed at physiological levels can be monitored. Typically, 2-20 time points can be recorded in the intact organism. Finally, we discuss how to extract the kinetic information on protein dynamics from two-color fluorescence recovery after photo-bleaching (FRAP) measurements and give advice how to keep the in vivo imager's five arch enemies--limited temporal and spatial resolution, injury of the animal, inactivation of proteins and movement artifacts--in check. While we focus on synapses, as model structure, the protocol can easily be adapted to study other developmental processes such as muscle growth, gut development or tracheal branching.
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.
PP2A and GSK-3beta Act Antagonistically to Regulate Active Zone Development
The synapse is composed of an active zone apposed to a postsynaptic cluster of neurotransmitter receptors. Each Drosophila neuromuscular junction comprises hundreds of such individual release sites apposed to clusters of glutamate receptors. Here, we show that protein phosphatase 2A (PP2A) is required for the development of structurally normal active zones opposite glutamate receptors. When PP2A is inhibited presynaptically, many glutamate receptor clusters are unapposed to Bruchpilot (Brp), an active zone protein required for normal transmitter release. These unapposed receptors are not due to presynaptic retraction of synaptic boutons, since other presynaptic components are still apposed to the entire postsynaptic specialization. Instead, these data suggest that Brp localization is regulated at the level of individual release sites. Live imaging of glutamate receptors demonstrates that this disruption to active zone development is accompanied by abnormal postsynaptic development, with decreased formation of glutamate receptor clusters. Remarkably, inhibition of the serine-threonine kinase GSK-3beta completely suppresses the active zone defect, as well as other synaptic morphology phenotypes associated with inhibition of PP2A. These data suggest that PP2A and GSK-3beta function antagonistically to control active zone development, providing a potential mechanism for regulating synaptic efficacy at a single release site.
Knockdown of Transactive Response DNA-binding Protein (TDP-43) Downregulates Histone Deacetylase 6
TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.
The Morphogene AmiC2 is Pivotal for Multicellular Development in the Cyanobacterium Nostoc Punctiforme
Filamentous cyanobacteria of the order Nostocales are primordial multicellular organisms, a property widely considered unique to eukaryotes. Their filaments are composed of hundreds of mutually dependent vegetative cells and regularly spaced N(2)-fixing heterocysts, exchanging metabolites and signalling molecules. Furthermore, they may differentiate specialized spore-like cells and motile filaments. However, the structural basis for cellular communication within the filament remained elusive. Here we present that mutation of a single gene, encoding cell wall amidase AmiC2, completely changes the morphology and abrogates cell differentiation and intercellular communication. Ultrastructural analysis revealed for the first time a contiguous peptidoglycan sacculus with individual cells connected by a single-layered septal cross-wall. The mutant forms irregular clusters of twisted cells connected by aberrant septa. Rapid intercellular molecule exchange takes place in wild-type filaments, but is completely abolished in the mutant, and this blockage obstructs any cell differentiation, indicating a fundamental importance of intercellular communication for cell differentiation in Nostoc. AmiC2-GFP localizes in the cell wall with a focus in the cross walls of dividing cells, implying that AmiC2 processes the newly synthesized septum into a functional cell-cell communication structure during cell division. AmiC2 thus can be considered as a novel morphogene required for cell-cell communication, cellular development and multicellularity.
