Richard W. Ordway

Active Zone Localization of Presynaptic Calcium Channels Encoded by the cacophony Locus of Drosophila

Presynaptic calcium channels play a central role in chemical synaptic transmission by providing the calcium trigger for evoked neurotransmitter release. These voltage-gated calcium channels are composed of a primary structural subunit, α1, as well as auxiliary β and α2δ subunits. Our previous genetic, molecular, and functional analysis has shown that the cacophony (cac) gene encodes a primary presynaptic calcium channel α1 subunit in Drosophila. Here we report that transgenic expression of a cac-encoded α1 subunit fused with enhanced green fluorescent protein efficiently rescues cac lethal mutations and allows in vivo analysis of calcium channel localization at active zones. The results reported here further characterize the primary role of cac-encoded calcium channels in neurotransmitter release. In addition, these studies provide a unique genetic tool for live imaging of functional presynaptic calcium channels in vivo and define a molecular marker for immunolocalization of other presynaptic proteins relative to active zones. These findings are expected to facilitate additional analysis of synaptic development and function in this important model system.

Fast synaptic fatigue in shibire mutants reveals a rapid requirementfor dynamin in synaptic vesicle membrane trafficking

The GTPase dynamin is involved in endocytosis in many cell types, as first revealed by temperature-sensitive paralytic mutations in the Drosophila dynamin gene, shibire (shi), which disrupt synaptic vesicle endocytosis and deplete synaptic terminals of vesicles. Here we report that shi synapses exhibit a fast synaptic fatigue phenotype within 20 ms of repetitive stimulation, which cannot be explained by vesicle depletion, as we confirmed by electron microscopy. These results suggest that, in addition to its well-characterized role in synaptic vesicle recycling, dynamin may be required for short-term maintenance of the readily releasable pool of synaptic vesicles.

Activity-dependent interactions of NSF and SNAP at living synapses.

As core components of the neurotransmitter release apparatus, SNAREs, NSF and SNAPs mediate fusion of neurotransmitter-filled synaptic vesicles within specialized regions of the presynaptic plasma membrane known as active zones (AZs). The present study combines genetic approaches in Drosophila with biochemical and live-imaging methods to provide new insights into the in vivo behavior and interactions of NSF and SNAP in neurotransmitter release. This work employs a temperature-sensitive (TS) paralytic NSF mutant, comatose, to show that disruption of NSF function results in activity-dependent redistribution of NSF and SNAP to periactive zone (PAZ) regions of the presynaptic plasma membrane and accumulation of protein complexes containing SNAREs, NSF and SNAP. Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Recovery After Photobleaching (FRAP) studies in comatose revealed that NSF and SNAP exhibit activity-dependent binding to each other within living presynaptic terminals as well as distinctive interactions and mobilities. These observations extend current models describing the spatial organization of NSF, SNAP and SNARE proteins in synaptic vesicle trafficking.

A Tripartite Synapse Model in Drosophila

Tripartite (three-part) synapses are defined by physical and functional interactions of glia with pre- and post-synaptic elements. Although tripartite synapses are thought to be of widespread importance in neurological health and disease, we are only beginning to develop an understanding of glial contributions to synaptic function. In contrast to studies of neuronal mechanisms, a significant limitation has been the lack of an invertebrate genetic model system in which conserved mechanisms of tripartite synapse function may be examined through large-scale application of forward genetics and genome-wide genetic tools. Here we report a Drosophila tripartite synapse model which exhibits morphological and functional properties similar to those of mammalian synapses, including glial regulation of extracellular glutamate, synaptically-induced glial calcium transients and glial coupling of synapses with tracheal structures mediating gas exchange. In combination with classical and cell-type specific genetic approaches in Drosophila, this model is expected to provide new insights into the molecular and cellular mechanisms of tripartite synapse function.

MAP1 structural organization in Drosophila: in vivo analysis of FUTSCH reveals heavy- and light-chain subunits generated by proteolytic processing at a conserved cleavage site

The MAP1 (microtubule-associated protein 1) family is a class of microtubule-binding proteins represented by mammalian MAP1A, MAP1B and the recently identified MAP1S. MAP1A and MAP1B are expressed in the nervous system and thought to mediate interactions of the microtubule-based cytoskeleton in neural development and function. The characteristic structural organization of mammalian MAP1s, which are composed of heavy- and light-chain subunits, requires proteolytic cleavage of a precursor polypeptide encoded by the corresponding map1 gene. MAP1 function in Drosophila appears to be fulfilled by a single gene, futsch. Although the futsch gene product is known to share several important functional properties with mammalian MAP1s, whether it adopts the same basic structural organization has not been addressed. Here, we report the identification of a Drosophila MAP1 light chain, LC(f), produced by proteolytic cleavage of a futsch-encoded precursor polypeptide, and confirm co-localization and co-assembly of the heavy chain and LC(f) cleavage products. Furthermore, the in vivo properties of MAP1 proteins were further defined through precise MS identification of a conserved proteolytic cleavage site within the futsch-encoded MAP1 precursor and demonstration of light-chain diversity represented by multiple LC(f) variants. Taken together, these findings establish conservation of proteolytic processing and structural organization among mammalian and Drosophila MAP1 proteins and are expected to enhance genetic analysis of conserved MAP1 functions within the neuronal cytoskeleton.

Small heat shock proteins mediate cell-autonomous and -nonautonomous protection in a Drosophila model for environmental-stress-induced degeneration.

ABSTRACT Cell and tissue degeneration, and the development of degenerative diseases, are influenced by genetic and environmental factors that affect protein misfolding and proteotoxicity. To better understand the role of the environment in degeneration, we developed a genetic model for heat shock (HS)-stress-induced degeneration in Drosophila. This model exhibits a unique combination of features that enhance genetic analysis of degeneration and protection mechanisms involving environmental stress. These include cell-type-specific failure of proteostasis and degeneration in response to global stress, cell-nonautonomous interactions within a simple and accessible network of susceptible cell types, and precise temporal control over the induction of degeneration. In wild-type flies, HS stress causes selective loss of the flight ability and degeneration of three susceptible cell types comprising the flight motor: muscle, motor neurons and associated glia. Other motor behaviors persist and, accordingly, the corresponding cell types controlling leg motor function are resistant to degeneration. Flight motor degeneration was preceded by a failure of muscle proteostasis characterized by diffuse ubiquitinated protein aggregates. Moreover, muscle-specific overexpression of a small heat shock protein (HSP), HSP23, promoted proteostasis and protected muscle from HS stress. Notably, neurons and glia were protected as well, indicating that a small HSP can mediate cell-nonautonomous protection. Cell-autonomous protection of muscle was characterized by a distinct distribution of ubiquitinated proteins, including perinuclear localization and clearance of protein aggregates associated with the perinuclear microtubule network. This network was severely disrupted in wild-type preparations prior to degeneration, suggesting that it serves an important role in muscle proteostasis and protection. Finally, studies of resistant leg muscles revealed that they sustain proteostasis and the microtubule cytoskeleton after HS stress. These findings establish a model for genetic analysis of degeneration and protection mechanisms involving contributions of environmental factors, and advance our understanding of the protective functions and therapeutic potential of small HSPs. Summary: A Drosophila model for environmental-stress-induced degeneration exhibits key features for genetic analysis of degenerative disease mechanisms and reveals new forms of protection mediated by small heat shock proteins.

A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult

Previous studies of Drosophila flight muscle neuromuscular synapses have revealed their tripartite architecture and established an attractive experimental model for genetic analysis of glial function in synaptic transmission. Here we extend these findings by defining a new Drosophila glial cell type, designated peripheral perisynaptic glia (PPG), which resides in the periphery and interacts specifically with fine motor axon branches forming neuromuscular synapses. Identification and specific labeling of PPG was achieved through cell type-specific RNAi-mediated knockdown (KD) of a glial marker, Glutamine Synthetase 2 (GS2). In addition, comparison among different Drosophila neuromuscular synapse models from adult and larval developmental stages indicated the presence of tripartite synapses on several different muscle types in the adult. In contrast, PPG appear to be absent from larval body wall neuromuscular synapses, which do not exhibit a tripartite architecture but rather are imbedded in the muscle plasma membrane. Evolutionary conservation of tripartite synapse architecture and peripheral perisynaptic glia in vertebrates and Drosophila suggests ancient and conserved roles for glia-synapse interactions in synaptic transmission.