During illumination, the light-sensitive plasma membrane (rhabdomere) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition. However, the mechanism by which illumination is coupled to rhabdomere turnover remains unclear. We find that photoreceptors contain a light-dependent phospholipase D (PLD) activity. During illumination, loss of PLD resulted in an enhanced reduction in rhabdomere size, accumulation of Rab7 positive, rhodopsin1-containing vesicles (RLVs) in the cell body and reduced rhodopsin protein. These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD. In wild-type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function. Thus, during illumination, PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition.DOI: http://dx.doi.org/10.7554/eLife.18515.001

Phosphatidic acid (PA) is a simple glycerophospholipid with a well-established role as an intermediate in phospholipid biosynthesis. In addition to its role in lipid biosynthesis, PA has been proposed to act as a signaling molecule that modulates several aspects of cell biology including membrane transport. PA can be generated in eukaryotic cells by several enzymes whose activity is regulated in the context of signal transduction and enzymes that can metabolize PA thus terminating its signaling activity have also been described. Further, several studies have identified PA binding proteins and changes in their activity are proposed to be mediators of the signaling activity of this lipid. Together these enzymes and proteins constitute a PA signaling toolkit that mediates the signaling functions of PA in cells. Recently, a number of novel genetic models for the analysis of PA function in vivo and analytical methods to quantify PA levels in cells have been developed and promise to enhance our understanding of PA functions. Studies of several elements of the PA signaling toolkit in a single cell type have been performed and are presented to provide a perspective on our understanding of the biochemical and functional organization of pools of PA in a eukaryotic cell. Finally, we also provide a perspective on the potential role of PA in human disease, synthesizing studies from model organisms, human disease genetics and analysis using recently developed PLD inhibitors.

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to s 2 -adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the s 2 -adaptin mutation. Genetic perturbation of s 2 -adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor b (TGFb) signaling was altered in these mutants and was restored by normalizing s 2 -adaptin in neurons. Thus, our data suggest that (1) while s 2 -adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during highfrequency nerve stimulation, and (2) s 2 -adaptin regulates NMJ morphology by attenuating TGFb signaling. KEYWORDS Drosophila; angur; synapse; physiology; pMAD S YNAPTIC transmission requires fusion of synaptic vesicles (SVs) at the active zones followed by their efficient retrieval and recycling through endocytic mechanisms (Heuser and Reese 1973;Jahn and Sudhof 1994). Retrieval and sorting of membrane lipids and vesicular proteins at the synapse are mediated by a well-orchestrated and coordinated action of several adapter and endocytic proteins (Stimson et al. 2001;Rikhy et al. 2002;Verstreken et al. 2002;Koh et al. 2004;Marie et al. 2004). Clathrin-mediated endocytosis (CME) is the primary pathway operative at the synapses for membrane retrieval (Granseth et al. 2006(Granseth et al. , 2007Heerssen et al. 2008;Dittman and Ryan 2009;McMahon and Boucrot 2011;Saheki and De Camilli 2012). Genetic analysis of the components of the CME pathway in Caenorhabditis elegans and Drosophila has revealed that this pathway is required for SV re-formation, and in many cases, blocking CME at synapses results in temperature-sensitive paralysis (Gonzalez-Gaitan and Jackle 1997;Zhang et al. 1998;Stimson et al. 2001;Koh et al. 2004Koh et al. , 2007Sato et al. 2009). Additionally, CME plays a crucial role in regulating synaptic morphology (Rikhy et al. 2002;Koh et al. 2004Koh et al. , 2007Dickman et al. 2006). At Drosophila NMJs, blocking CME results in enhanced bone morphogenetic protein (BMP) si...

Phosphatidylinositol transfer proteins (PITPs) are essential regulators of PLC signalling. The PI transfer domain (PITPd) of multi-domain PITPs is reported to be sufficient for function, questioning the relevance of other domains in the protein. In photoreceptors, loss of RDGBα, a multi-domain PITP localized to membrane contact sites (MCSs), results in multiple defects during PLC signalling. Here, we report that the PITPd of RDGBα does not localize to MCSs and fails to support function during strong PLC stimulation. We show that the MCS localization of RDGBα depends on the interaction of its FFAT motif with dVAP-A. Disruption of the FFAT motif (RDGB) or downregulation of dVAP-A, both result in mis-localization of RDGBα and are associated with loss of function. Importantly, the ability of the PITPd in full-length RDGB to rescue mutant phenotypes was significantly worse than that of the PITPd alone, indicating that an intact FFAT motif is necessary for PITPd activity Thus, the interaction between the FFAT motif and dVAP-A confers not only localization but also intramolecular regulation on lipid transfer by the PITPd of RDGBα. This article has an associated First Person interview with the first author of the paper.