SummaryMost cells store neutral lipids in a dedicated compartment, the lipid droplet (LD). These LDs are structurally and functionally conserved across species. In higher eukaryotes, LDs are covered by abundant scaffolding proteins, such as the oleosins in plants and perilipins (PLINs) in animal cells. Saccharomyces cerevisiae, however, has no homologues of these scaffolding proteins. To analyze a possible function of these proteins in the biogenesis of LDs, oleosin and perilipin family members (PLIN1, ADRP/PLIN2 and TIP47/PLIN3) were expressed in yeast cells and their targeting to LDs, membrane association and function in neutral lipid homeostasis and LD biogenesis were analyzed. When expressed in wild-type cells, these proteins were properly targeted to LDs. However, when expressed in cells lacking LDs, oleosin was localized to the ER bilayer and was rapidly degraded. PLINs, on the other hand, did not localize to the ER membrane in the absence of LDs and lost their membrane association. Photobleaching experiments revealed that PLIN2 and PLIN3 rapidly exchanged their LD association, but PLINs did not move as quickly as integral membrane proteins, such as oleosin, over the LD surface. Interestingly, expression of these scaffolding LD proteins in mutant cells containing elevated levels of neutral lipids within the ER bilayer resulted in the formation of LDs. These results suggest that these LD scaffolding proteins promote the sequestration of neutral lipids from the ER bilayer and thereby induce LD formation. Consistent with this proposition, addition of a cell-permeable diacylglycerol (DAG) was sufficient to promote LD formation in cells expressing the LD scaffolding proteins but lacking the capacity to synthesize storage lipids.

Lipid droplets are found in most organisms where they serve to store energy in the form of neutral lipids. They are formed at the endoplasmic reticulum (ER) membrane where the neutral-lipidsynthesizing enzymes are located. Recent results indicate that lipid droplets remain functionally connected to the ER membrane in yeast and mammalian cells to allow the exchange of both lipids and integral membrane proteins between the two compartments. The precise nature of the interface between the ER membrane and lipid droplets, however, is still ill-defined. Here, we probe the topology of lipid droplet biogenesis by artificially targeting proteins that have high affinity for lipid droplets to inside the luminal compartment of the ER. Unexpectedly, these proteins still localize to lipid droplets in both yeast and mammalian cells, indicating that lipid droplets are accessible from within the ER lumen. These data are consistent with a model in which lipid droplets form a specialized domain in the ER membrane that is accessible from both the cytosolic and the ER luminal side.

Inter‐organelle communication between closely apposed membranes is proposed at membrane contact sites (MCS). However, the regulation of MCS structure and their functional relevance in vivo remain debated. The extended synaptotagmins (Esyt) are evolutionarily conserved proteins proposed to function at MCS. However, loss of all three Esyts in yeast or mammals shows minimal phenotypes questioning the functional importance of Esyt. We report that in Drosophila photoreceptors, MCS number is regulated by PLCβ activity. Photoreceptors of a null allele of Drosophila extended synaptotagmin (dEsyt) show loss of ER‐PM MCS. Loss of dEsyt results in mislocalization of RDGB, an MCS localized lipid transfer protein, required for photoreceptor structure and function, ultimately leading to retinal degeneration. dEsyt depletion enhanced the retinal degeneration, reduced light responses and slower rates of plasma membrane PIP2 resynthesis seen in rdgB mutants. Thus, dEsyt function and PLCβ signaling regulate ER‐PM MCS structure and lipid transfer in Drosophila photoreceptors.

SummaryInter-organelle communication between closely apposed membranes is proposed at Membrane Contact Sites (MCS). However the regulation of MCS structure and their functional relevance in vivo remain debated. The extended synaptotagmins (Esyt) are evolutionarily conserved proteins proposed to function at MCS. However, loss of all three Esyts in yeast or mammals shows minimal phenotypes questioning the functional importance of Esyt. We report that in Drosophila photoreceptors, MCS number is regulated by PLCβ activity. Photoreceptors of a null allele of Drosophila extended synaptotagmin (dEsyt) show loss of ER-PM MCS. Loss of dEsyt results in mislocalization of RDGB, an MCS localized lipid transfer protein, required for photoreceptor structure and function, ultimately leading to retinal degeneration. dEsyt depletion enhanced the retinal degeneration, reduced light responses and slower rates of plasma membrane PIP2 resynthesis seen in rdgB mutants. Thus, dEsyt function and PLCβ signaling regulate ER-PM MCS structure and lipid transfer in Drosophila photoreceptors.

Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remains poorly understood. Here we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with BMP type I receptor, Thickveins (Tkv), and Daughter against decapentaplegic (Dad), two of the components of BMP signaling. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the NMJ and results in a significant reduction in Tkv-positive early endosomes in the presynaptic terminals. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. Consistent with the role of σ2-adaptin and Rab11 in the regulation of the same signaling pathway, a mutation in Rab11 or overexpression of a GDP-locked form of Rab11 (Rab11S25N) phenocopies the morphological and signaling defects of the σ2-adaptin mutants. Finally, we demonstrate that σ2-adaptin mutants show an accumulation of large vesicles and massive membranous structures, akin to endosomes at the synapse. Thus, we propose a model in which AP2 regulates Tkv internalization and recycling through a process that requires Rab11 activity to control the synaptic growth.