Extended synaptotagmin regulates membrane contact site structure and lipid transfer function in vivo.

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 PLCß 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 PLCß signaling regulate ER-PM MCS structure and lipid transfer in Drosophila photoreceptors.

Mature lipid droplets are accessible to ER luminal proteins.

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-lipid-synthesizing 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.

Expression of oleosin and perilipins in yeast promotes formation of lipid droplets from the endoplasmic reticulum.

Most 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.

A genetic screen to uncover mechanisms underlying lipid transfer protein function at membrane contact sites.

Lipid transfer proteins mediate the transfer of lipids between organelle membranes, and the loss of function of these proteins has been linked to neurodegeneration. However, the mechanism by which loss of lipid transfer activity leads to neurodegeneration is not understood. In Drosophila photoreceptors, depletion of retinal degeneration B (RDGB), a phosphatidylinositol transfer protein, leads to defective phototransduction and retinal degeneration, but the mechanism by which loss of this activity leads to retinal degeneration is not understood. RDGB is localized to membrane contact sites through the interaction of its FFAT motif with the ER integral protein VAP. To identify regulators of RDGB function in vivo, we depleted more than 300 VAP-interacting proteins and identified a set of 52 suppressors of rdgB The molecular identity of these suppressors indicates a role of novel lipids in regulating RDGB function and of transcriptional and ubiquitination processes in mediating retinal degeneration in rdgB9 The human homologs of several of these molecules have been implicated in neurodevelopmental diseases underscoring the importance of VAP-mediated processes in these disorders.

Structural organization of RDGB (retinal degeneration B), a multi-domain lipid transfer protein: a molecular modelling and simulation based approach.

Lipid transfer proteins (LTPs) that shuttle lipids at membrane contact sites (MCS) play an important role in maintaining cellular homeostasis. One such important LTP is the Retinal Degeneration B (RDGB) protein. RDGB is localized at the MCS formed between the endoplasmic reticulum (ER) and the apical plasma membrane (PM) in Drosophila photoreceptors where it transfers phosphatidylinositol (PI) during G-protein coupled phospholipase C signalling. Previously, the C-terminal domains of RDGB have been shown to be essential for its function and accurate localization. In this study, using in-silico integrative modelling we predict the structure of entire RDGB protein in complex with the ER membrane protein VAP. The structure of RDGB has then been used to decipher the structural features of the protein important for its orientation at the contact site. Using this structure, we identify two lysine residues in the C-terminal helix of the LNS2 domain important for interaction with the PM. Using molecular docking, we also identify an unstructured region USR1, immediately c-terminal to the PITP domain that is important for the interaction of RDGB with VAP. Overall the 10.06 nm length of the predicted RDGB-VAP complex spans the distance between the PM and ER and is consistent with the cytoplasmic gap between the ER and PM measured by transmission electron microscopy in photoreceptors. Overall our model explains the topology of the RDGB-VAP complex at this ER-PM contact site and paves the way for analysis of lipid transfer function in this setting.Communicated by Ramaswamy H. Sarma.

A Comparative Study of Solifenacin, Mirabegron, and Their Combination as Bladder Relaxants in the Management of Overactive Bladder.

Introduction  Overactive bladder (OAB) is a medical state that presents as the urgency of urine and increased frequency of micturition and is diagnosed on the basis of the presence of these symptoms in the absence of other explainable diagnoses. The management of this condition includes conservative management, medical management/pharmacotherapy, and surgical management. The overactive bladder has been treated with smooth muscle relaxants, but there are conflicting results. Hence, this study aimed to assess the result of the two smooth muscle relaxants, mirabegron and solifenacin, and their combination to manage an overactive bladder. Methodology  A clinical trial was conducted at Swaroop Rani Nehru Hospital, Motilal Nehru Medical College, Prayagraj, India, over the period from November 2019 to December 2020. Ninety patients with OAB were divided into three groups: G1, G2, and G3. These groups were administered solifenacin, mirabegron, and a combination of mirabegron and solifenacin (S+M), respectively. Follow-ups were conducted at 2, 4, 12, and 18 weeks for evaluation. Data were entered into IBM SPSS Statistics for Windows, Version 23 (Released 2015; IBM Corp., Armonk, New York, United States). Appropriate statistical tests, including the chi-square and ANOVA, were employed in this study. Observation  The combination of mirabegron and solifenacin was significantly more effective in terms of response compared to solifenacin alone. There was no significant difference between solifenacin versus mirabegron, or between mirabegron (M) and the combination of mirabegron (M) and solifenacin (S). Side effects were more severe in patients taking high doses of solifenacin. Conclusion  The S + M combination has higher efficacy than solifenacin and mirabegron when given alone.

AP2 Regulates Thickveins Trafficking to Attenuate NMJ Growth Signaling in Drosophila.

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 remain poorly understood. Here, we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila Ultrastructural analysis of σ2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of σ2-adaptin mutations. We propose a model in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth.

Expression of perilipin 5 promotes lipid droplet formation in yeast.

Neutral lipids are packed into dedicated intracellular compartments termed lipid droplets (LDs). LDs are spherical structures delineated by an unusual lipid monolayer and they harbor a specific set of proteins, many of which function in lipid synthesis and lipid turnover. In mammals, LDs are covered by abundant scaffolding proteins, the perilipins (PLIN1-5). LDs in yeast are functionally similar to that of mammalian cells, but they lack the perilipins. We have previously shown that perilipins (PLIN1-3) are properly targeted to LDs when expressed in yeast and that they promote LD formation from the ER membrane enriched in neutral lipids. Here we address the question whether PLIN5 (OXPAT) has a similar function. Both human and murine PLIN5 were properly targeted to yeast LDs, but the protein localized to the cytosol and its steady-state level was reduced when expressed in yeast mutants lacking the capacity to synthesize storage lipids. When expressed in cells containing high levels of neutral lipids within the membrane of the endoplasmatic reticulum, PLIN5 promoted the formation of LDs. Interestingly, PLIN5 was properly targeted to LDs, irrespective of whether these LDs were filled with triacylglycerol or steryl esters, indicating that PLIN5 did not exhibit targeting specificity for a particular subtypes of LDs as was reported for mammalian cells.