SUMMARY Rapid neurotransmitter release depends on the Ca2+-sensor Synaptotagmin-1 and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Synaptotagmin-1 triggers release remains unclear, in part because elucidating high-resolution structures of Synaptotagmin-1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode where basic residues in the concave side of the Synaptotagmin-1 C2B domain β-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Synaptotagmin-1 that markedly impair SNARE-complex binding in vitro and Synaptotagmin-1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Synaptotagmin-1 function. Our results support a model whereby their dynamic interaction facilitates cooperation between synaptotagmin-1 and the SNAREs in inducing membrane fusion.

SUMMARY Obese individuals exhibit an increase in pancreatic β-cell mass; conversely, scarce nutrition during pregnancy has been linked to β-cell insufficiency in the offspring (reviewed in [1, 2]). These phenomena are thought to be mediated mainly through effects on β-cell proliferation, since a nutrient sensitive β-cell progenitor population in the pancreas has not been identified. Here, we employed the FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator) system to investigate β-cell replication in real-time, and found that high nutrient concentrations induce rapid β-cell proliferation. Importantly, we found that high nutrient concentrations also stimulate β-cell differentiation from progenitors in the intrapancreatic duct (IPD). Using a new zebrafish line where β-cells are constitutively ablated, we further show that β-cell loss and high nutrient intake synergistically activate these progenitors. At the cellular level, this activation process causes ductal cell reorganization as it stimulates their proliferation and differentiation. Notably, we link the nutrient-dependent activation of these progenitors to a down-regulation of Notch signaling specifically within the IPD. Furthermore, we show that the nutrient sensor mechanistic Target Of Rapamycin (mTOR) is required for endocrine differentiation from the IPD under physiological conditions as well as in the diabetic state. This study thus reveals critical insights into how cells modulate their plasticity in response to metabolic cues and identifies nutrient sensitive progenitors in the mature pancreas.

SUMMARY Contacts between the endoplasmic reticulum and the plasma membrane involve extended synaptotagmins (E-Syts) in mammals or tricalbins in yeast, proteins with multiple C2 domains. One of the tandem C2 domains of E-Syt2 is predicted to bind Ca2+, but no Ca2+-dependent function has been attributed to this protein. We have determined the crystal structures of the tandem C2 domains of E-Syt2 in the absence and presence of Ca2+, and analyzed their Ca2+-binding properties by NMR spectroscopy. Our data reveal an unexpected V-shaped structure with a rigid orientation between the two C2 domains that is not substantially altered by Ca2+. The E-Syt2 C2A domain binds up to four Ca2+ ions, whereas the C2B domain does not bind Ca2+. These results suggest that E-Syt2 performs an as yet unidentified Ca2+-dependent function through its C2A domain, and uncover fundamental differences between the properties of the tandem C2 domains of E-Syts and synaptotagmins.

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is an important membrane transporter that contributes to cellular disposition of many endobiotic and xenobiotic agents, and it can also confer multidrug resistance. This study aimed to investigate the role of human noncoding microRNA-1291 (hsa-miR-1291) in regulation of ABCC1 and drug disposition. Bioinformatics analyses indicated that hsa-miR-1291, localized within the small nucleolar RNA H/ACA box 34 (SNORA34), might target ABCC1 39-untranslated region (39UTR). Using splinted ligation small RNA detection method, we found that SNORA34 was processed into hsa-miR-1291 in human pancreatic carcinoma PANC-1 cells. Luciferase reporter assays showed that ABCC1 39-UTRluciferase activity was decreased by 20% in cells transfected with hsa-miR-1291 expression plasmid, and increased by 40% in cells transfected with hsa-miR-1291 antagomir. Furthermore, immunoblot study revealed that ABCC1 protein expression was sharply reduced in hsa-miR-1291-stably transfected PANC-1 cells, which was attenuated by hsa-miR-1291 antagomir. The change of ABCC1 protein expression was associated with an alternation in mRNA expression. In addition, hsa-miR-1291-directed downregulation of ABCC1 led to a greater intracellular drug accumulation and sensitized the cells to doxorubicin. Together, our results indicate that hsa-miR-1291 is derived from SNORA34 and modulates cellular drug disposition and chemosensitivity through regulation of ABCC1 expression. These findings shall improve the understanding of microRNAcontrolled epigenetic regulatory mechanisms underlying multidrug resistance and interindividual variability in pharmacokinetics.