Fife, aDrosophilaPiccolo-RIM Homolog, Promotes Active Zone Organization and Neurotransmitter Release

Abstract

Neuronal communication depends on the precisely orchestrated release of neurotransmitter at specialized sites called active zones (AZs). A small number of scaffolding and cytoskeletal proteins comprising the cytomatrix of the active zone (CAZ) are thought to organize the architecture and functional properties of AZs. The majority of CAZ proteins are evolutionarily conserved, underscoring the fundamental similarities in neurotransmission at all synapses. However, core CAZ proteins Piccolo and Bassoon have long been believed exclusive to vertebrates, raising intriguing questions about the conservation of the molecular mechanisms that regulate presynaptic properties. Here, we present the identification of a piccolo-rim-related gene in invertebrates, together with molecular phylogenetic analyses that indicate the encoded proteins may represent Piccolo orthologs. In accordance, we find that the Drosophila homolog, Fife, is neuronal and localizes to presynaptic AZs. To investigate the in vivo function of Fife, we generated a deletion of the fife locus. We find that evoked neurotransmitter release is substantially decreased in fife mutants and loss of fife results in motor deficits. Through morphological analysis of fife synapses, we identify underlying AZ abnormalities including pervasive presynaptic membrane detachments and reduced synaptic vesicle clustering. Our data demonstrate the conservation of a Piccolo-related protein in invertebrates and identify critical roles for Fife in regulating AZ structure and function. These findings suggest the CAZ is more conserved than previously thought, and open the door to a more complete understanding of how CAZ proteins regulate presynaptic structure and function through genetic studies in simpler model systems.

Transmission, Development, and Plasticity of Synapses

“…A Drosophila ortholog of Piccolo, a core CAZ protein previously thought to exist only in vertebrates, was recently identified and named fife. Fife colocalizes with Brp at AZs, and loss of fife results in defects in AZ ultrastructure, including detachments of the presynaptic membrane and a reduction of SV clustering at T-bars (Bruckner et al 2012). Another recently identified CAZ component is the presynaptic scaffolding protein Dyschronic (DYSC).…”

Presynaptic active zones in invertebrates and vertebrates

“…However, studies by Bruckner et al [24] have recently identified a Piccolo homolog, aptly named Fife, which is selectively localized at Drosophila AZs. Furthermore, the detached membrane phenotype implies a role in transsynaptic adhesion [24]. Fife loss-of-function has profound effects on AZ organization and synaptic transmission, including a dramatic decrease in excitatory junctional potential amplitude and quantal content.…”

“…Two exceptions are Piccolo and Bassoon, which may have evolved to perform vertebrate-specific functions. However, studies by Bruckner et al [24] have recently identified a Piccolo homolog, aptly named Fife, which is selectively localized at Drosophila AZs. Structurally, Fife is a hybrid molecule with features from both Piccolo and RIM, including zinc finger (ZnF), PDZ, and C2 domains.…”

“…Intriguingly, floating T-bars are occasionally seen at fly NMJs lacking RBP [21], suggesting that Fife and RBP both function to tether SVs at the AZ. Furthermore, the detached membrane phenotype implies a role in transsynaptic adhesion [24]. Interestingly, several CAZ proteins have been identified to work in concert with the transsynaptic adhesion/signaling molecules Neurexin-1 (Nrx-1) and Neuroligin-1 (Nlg1) at the Drosophila NMJ, including Syd1, Syd2/DLiprina, and Wrd (a regulatory subunit B' of the phosphatase PP2A).…”

The actin cytoskeleton in presynaptic assembly

“…89 Originally thought to be unique to vertebrates, a Piccolo homolog, Fife, was identified in invertebrates. 91 Knockdown of Piccolo with shRNA causes dispersion of Synapsin1a and F-actin at the presynaptic terminal and causes a loss of tight association between Synapsin1a, actin, and synaptic vesicles. 56,92 Piccolo also interacts with actin regulators such as profilin.…”