A long-standing quest in the autophagy field is to define the membrane origin of the autophagosome. We have established a cell-free assay based on LC3 lipidation that recapitulates multiple regulatory hallmarks of early autophagosome biogenesis. Using a systematic membrane fractionation approach, we have identified the ER-Golgi intermediate compartment (ERGIC) as the most efficient membrane substrate for LC3 lipidation. Further studies indicate that the ERGIC plays an essential role to trigger LC3 lipidation and autophagosome biogenesis by recruiting the key early autophagic factor ATG14.

Formation of the autophagosome requires significant membrane input from cellular organelles. However, no direct evidence has been developed to link autophagic factors and the mobilization of membranes to generate the phagophore. Previously, we established a cell-free LC3 lipidation reaction to identify the ER-Golgi intermediate compartment (ERGIC) as a membrane source for LC3 lipidation, a key step of autophagosome biogenesis (Ge et al., eLife 2013; 2:e00947). We now report that starvation activation of autophagic phosphotidylinositol-3 kinase (PI3K) induces the generation of small vesicles active in LC3 lipidation. Subcellular fractionation studies identified the ERGIC as the donor membrane in the generation of small lipidation-active vesicles. COPII proteins are recruited to the ERGIC membrane in starved cells, dependent on active PI3K. We conclude that starvation activates the autophagic PI3K, which in turn induces the recruitment of COPII to the ERGIC to bud LC3 lipidation-active vesicles as one potential membrane source of the autophagosome.

Autophagosome biogenesis requires efficient mobilization and delivery of membranes from intracellular sources. How these membranes are mobilized remains poorly understood. Our recent work reported an autophagic signal-induced membrane mobilization event from the ER-Golgi intermediate compartment (ERGIC) to generate an early autophagosomal membrane precursor. We found that starvation activates the autophagic phosphatidylinositol 3-kinase, which promotes a relocation of COPII proteins from the ER-exit sites to the ERGIC. The relocation of COPII generates ERGIC-derived COPII vesicles as a membrane template for LC3 lipidation, a key step for autophagosome biogenesis.

Formation of the autophagosome requires significant membrane input from cellular organelles. However, no direct evidence has been developed to link autophagic factors and the mobilization of membranes to generate the phagophore. Previously, we established a cell-free LC3 lipidation reaction to identify the ER-Golgi intermediate compartment (ERGIC) as a membrane source for LC3 lipidation, a key step of autophagosome biogenesis (Ge et al., eLife 2013; 2:e00947). We now report that starvation activation of autophagic phosphotidylinositol-3 kinase (PI3K) induces the generation of small vesicles active in LC3 lipidation. Subcellular fractionation studies identified the ERGIC as the donor membrane in the generation of small lipidation-active vesicles. COPII proteins are recruited to the ERGIC membrane in starved cells, dependent on active PI3K. We conclude that starvation activates the autophagic PI3K, which in turn induces the recruitment of COPII to the ERGIC to bud LC3 lipidation-active vesicles as one potential membrane source of the autophagosome.

Thromboembolic complications (TECs) of bileaflet mechanical heart valves (BMHVs) are believed to be due to the nonphysiologic mechanical stresses imposed on blood elements by the hinge flows. Relating hinge flow features to design features is, therefore, essential to ultimately design BMHVs with lower TEC rates. This study aims at simulating the pulsatile three-dimensional hinge flows of three BMHVs and estimating the TEC potential associated with each hinge design. Hinge geometries are constructed from micro-computed tomography scans of BMHVs. Simulations are conducted using a Cartesian sharp-interface immersed-boundary methodology combined with a second-order accurate fractional-step method. Leaflet motion and flow boundary conditions are extracted from fluid–structure-interaction simulations of BMHV bulk flow. The numerical results are analyzed using a particle-tracking approach coupled with existing blood damage models. The gap width and, more importantly, the shape of the recess and leaflet are found to impact the flow distribution and TEC potential. Smooth, streamlined surfaces appear to be more favorable than sharp corners or sudden shape transitions. The developed framework will enable pragmatic and cost-efficient preclinical evaluation of BMHV prototypes prior to valve manufacturing. Application to a wide range of hinges with varying design parameters will eventually help in determining the optimal hinge design.

Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes. DOI:http://dx.doi.org/10.7554/eLife.00947.001.

Thromboembolic complications of bileaflet mechanical heart valves (BMHV) are believed to be due to detrimental stresses imposed on blood elements by the hinge flows. Characterization of these flows is thus crucial to identify the underlying causes for complications. In this study, we conduct three-dimensional pulsatile flow simulations through the hinge of a BMHV under aortic conditions. Hinge and leaflet geometries are reconstructed from the Micro-Computed Tomography scans of a BMHV. Simulations are conducted using a Cartesian sharp-interface immersed-boundary methodology combined with a second-order accurate fractional-step method. Physiologic flow boundary conditions and leaflet motion are extracted from the Fluid–Structure Interaction simulations of the bulk of the flow through a BMHV. Calculations reveal the presence, throughout the cardiac cycle, of flow patterns known to be detrimental to blood elements. Flow fields are characterized by: (1) complex systolic flows, with rotating structures and slow reverse flow pattern, and (2) two strong diastolic leakage jets accompanied by fast reverse flow at the hinge bottom. Elevated shear stresses, up to 1920 dyn/cm2 during systole and 6115 dyn/cm2 during diastole, are reported. This study underscores the need to conduct three-dimensional simulations throughout the cardiac cycle to fully characterize the complexity and thromboembolic potential of the hinge flows.

The biogenesis of autophagosomes entails the nucleation and growth of a double-membrane sheet, the phagophore, which engulfs cytosol for delivery to the lysosome. Genetic studies have identified a class of Atg proteins that are essential for the process, yet the molecular mechanism of autophagosome biogenesis has been elusive. Proteomic, structural, super-resolution imaging, and biochemical reconstitution experiments have begun to fill in some of the gaps. This review describes progress and prospects for obtaining a four-dimensional network model of the nucleation and growth of the phagophore.

Background

Equipoise of transcatheter aortic valve replacement with surgical aortic valve replacement in intermediate-risk patients has been demonstrated. As transcatheter aortic valve replacement usage expands, questions regarding long-term durability become paramount. Valve design impacts durability with regions of increased leaflet stress being vulnerable to early failure. However, transcatheter aortic valve (TAV) leaflet stresses are unknown. The objective of this study was to determine stent and leaflet stresses of second-generation balloon-expandable TAV.

Methods

Commercial 29-mm Edwards Sapien XT (Edwards Lifesciences, Irvine, CA) valves underwent high-resolution microcomputed tomography scanning to develop precise three-dimensional geometric mesh. Compressed and uncompressed TAVs were modeled under systemic pressure using finite element software. Material properties of stent were based on cobalt-chromium, whereas those for leaflets were obtained from surgical bioprostheses.

Results

Maximum and minimum principal stresses on uncompressed Sapien XT TAV were 1.63 MPa and -0.36 MPa on leaflets and 93.3 MPa and -105.6 MPa on stent at diastolic pressure. Peak leaflet stress was observed at commissural tips where leaflets connected to the stent. For compressed TAV to 26 mm, maximum and minimum principal stresses were 1.55 MPa and -0.63 MPa on leaflets and 526.1 MPa and -902.2 MPa on stent at diastolic pressure. Peak leaflet stress was located at similar position and also along the suture line with the Dacron (C. R. Bard, Haverhill, PA).

Conclusions

Stress analysis of two extreme deployed geometries of 29-mm Edwards Sapien XT using exact geometry from high-resolution scans demonstrated that peak stresses for TAV leaflets were present at commissural tips where leaflets were attached. These regions would be mostly likely to initiate degeneration.

Valvular hemolysis and thrombosis are common complications associated with stenotic heart valves. This study aims to determine the extent to which hemodynamics induce such traumatic events. The viscous shear stress downstream of a severely calcified bioprosthetic valve was evaluated via in vitro 2D particle image velocimetry measurements. The blood cell membrane response to the measured stresses was then quantified using 3D immersed-boundary computational simulations. The shear stress level at the boundary layer of the jet flow formed downstream of the valve orifice was observed to reach a maximum of 1000-1700 dyn/cm(2), which was beyond the threshold values reported for platelet activation (100-1000 dyn/cm(2)) and within the range of thresholds reported for red blood cell (RBC) damage (1000-2000 dyn/cm(2)). Computational simulations demonstrated that the resultant tensions at the RBC membrane surface were unlikely to cause instant rupture, but likely to lead to membrane plastic failure. The resultant tensions at the platelet surface were also calculated and the potential damage was discussed. It was concluded that although shear-induced thrombotic trauma is very likely in stenotic heart valves, instant hemolysis is unlikely and the shear-induced damage to RBCs is mostly subhemolytic.