RNA interference-mediated knockdown of a GATA factor reveals a link to anautogeny in the mosquito Aedes aegypti

Blood feeding tightly regulates the reproductive cycle in anautogenous mosquitoes. Vitellogenesis (the synthesis of yolk protein precursors) is a key event in the mosquito reproductive cycle and is activated in response to a blood meal. Before blood feeding, Aedes aegypti is in a state of reproductive arrest during which the yolk protein precursor genes (YPPs) are repressed. The regulatory region of the major YPP gene vitellogenin (Vg) has multiple GATA-binding sites required for the high expression level of this gene. However, a GATA factor (AaGATAr) likely acts as a repressor, preventing activation of this gene before a blood meal. Here we report in vivo data confirming the role of AaGATAr as a repressor of the Vg gene at the state of previtellogenic arrest. Using an RNA interference (RNAi)-mediated technique in conjunction with the Sindbis viral expression system, we show that knockdown of the AaGATAr gene results in an increased basal level of expression of the Vg gene and an elevated response to the steroid hormone 20-hydroxyecdysone in mosquitoes in a state of arrest. These experiments have revealed a component in the molecular mechanism by which anautogeny is maintained in A. aegypti.     

Linkage analysis identifies a locus for plasma von Willebrand factor undetected by genome-wide association

The plasma glycoprotein von Willebrand factor (VWF) exhibits fivefold antigen level variation across the normal human population determined by both genetic and environmental factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis, myocardial infarction, and stroke. To identify additional genetic determinants of VWF antigen levels and to minimize the impact of age and illness-related environmental factors, we performed genome-wide association analysis in two young and healthy cohorts (n = 1,152 and n = 2,310) and identified signals at ABO (P < 7.9E-139) and VWF (P < 5.5E-16), consistent with previous reports. Additionally, linkage analysis based on sibling structure within the cohorts, identified significant signals at chromosome 2q12–2p13 (LOD score 5.3) and at the ABO locus on chromosome 9q34 (LOD score 2.9) that explained 19.2% and 24.5% of the variance in VWF levels, respectively. Given its strong effect, the linkage region on chromosome 2 could harbor a potentially important determinant of bleeding and thrombosis risk. The absence of a chromosome 2 association signal in this or previous association studies suggests a causative gene harboring many genetic variants that are individually rare, but in aggregate common. These results raise the possibility that similar loci could explain a significant portion of the “missing heritability” for other complex genetic traits.     

In situ structural analysis reveals membrane shape transitions during autophagosome formation

Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.

Macroautophagy (autophagy hereafter) is a key pathway to maintain cellular homeostasis. In this process, a de novo synthesized double-membrane vesicle, the autophagosome, engulfs cellular material in response to stress conditions (1). This culminates in autophagosome fusion with lysosomes (or the vacuole in yeast) to remove and recycle its cargo. Fluorescence microscopy has identified the hierarchical order of the autophagy machinery during autophagosome biogenesis (2, 3). In addition, many of the membrane intermediates have been visualized at low resolution with conventional electron microscopy (4–7). These and other methods have revealed that autophagy proceeds in several steps: (I) membrane nucleation, (II) growth of the cup-shaped phagophore, (III) closure, and (IV) fusion of the autophagosome with the lytic compartment (8). Meanwhile, pioneering genetic and biochemical studies have revealed key regulators of autophagosome biogenesis (8, 9). In yeast, nitrogen starvation triggers the first step of phagophore nucleation through assembly of the molecular machinery in the pre-autophagosomal structure (PAS) next to the vacuole (10). The phagophore is initially formed by fusion of few vesicles carrying the transmembrane protein Atg9 (11–13). It then grows both by fusion of vesicles (e.g., Atg9 or COPII vesicles (14)) and by lipid transfer from the endoplasmic reticulum (ER) through protein complexes such as Atg2/Atg18 (15). Membrane expansion is further driven by conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine in the phagophore membrane (16). During growth, the initial membrane disk assumes a characteristic cup shape, a transition that is likely driven by the highly curved and therefore energetically unfavorable phagophore rim (17). After closure and maturation, the resulting autophagosome fuses with the vacuole, releasing the inner vesicle—now called “autophagic body”—for degradation.Despite the importance of autophagy and the efforts in deciphering the molecular machinery underlying it (8), it is still unknown how membranes are organized and transformed on an ultrastructural level during autophagosome biogenesis. In situ cryo-electron tomography (cryo-ET) can reveal membrane structures directly in their native cellular environment (18, 19). Yet monitoring the formation of an organelle poses the challenge to capture a rare event with many intermediates along the process. To overcome these hurdles, we combined several strategies to dissect the formation of autophagosomes using cryo-ET: (I) stimulating their formation to increase the abundance of all species involved, (II) using mutants that accumulate intermediates that are naturally short lived, and (III) fluorescently labeling the autophagy machinery or its cargo to specifically target those structures during focused ion beam (FIB) milling and tomogram acquisition.Using this approach, we captured the major membrane structures in bulk autophagy within their native context and at high resolution. Our detailed data analysis provides important insights into the biophysics of autophagosome biogenesis. While we focus here on yeast autophagy, our study highlights the potential of correlative cryo-ET in analyzing short-lived cellular structures and provides a general template for studying the formation of organelles.