Cellular double-membrane organelles at different stages of their genesis.

<p>(A–D) electron microscopy micrographs, (E) schematic illustration of the sequence of shape changes, and (F) schematic cross section of the sheet with possible bending directions. (A) A growing double-membrane sheet during spore formation in <i>Schizosaccharomyces pombe</i>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032753#pone.0032753-Shimoda1" target="_blank">[2]</a>. (B) A cup-shaped phagophore with immunogold label (black spots) for the mammalian Atg8 homologue GATE16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032753#pone.0032753-Kabeya1" target="_blank">[51]</a>. (C) A closed autophagosome with the double membrane clearly visible <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032753#pone.0032753-YlaAnttila2" target="_blank">[50]</a>. (D) A freeze-fracture electron micrograph showing the smooth autophagosomal membrane. In the upper right corner a small, particle-rich endosome had fused with the autophagosome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032753#pone.0032753-Fengsrud1" target="_blank">[47]</a>; the smooth surface of the autophagosome away from the fusion area suggests the absence of a protein coat. All scale bars correspond to 0.5 µm. The electron microscopy images were adapted with permissions of the J. Cell Sci. and Elsevier. (E) Schematic illustrations of the shape transition from a double-membrane sheet to a double-membrane vesicle (cross sections shown). The solid line represents one bilayer. Geometrical parameters used in the main text are indicated in the first cartoon. The transition between the flat sheet and the vesicle can be reversible. The final step of generating the double-membrane vesicle requires irreversible fission. (F) Schematic cross sections of the sheet and cup-shape morphologies. Three different segments of the shapes are distinguished: lower segment (1, dashed blue), upper segment (2, solid green), and highly curved rim (3, solid red). The sheet (middle) is characterized by zero mean curvatures of the upper and lower segments, <i>M</i>₁ = <i>M</i>₂ = 0. When the sheet bends downwards (left), the mean curvature of the lower segment is negative, <i>M</i>₁<0, and that of the upper segment is positive, <i>M</i>₂>0. The situation is reversed when the sheet bends upwards (right).</p

Reduced bending energy of double-membrane shapes,

<p><b>, as a function of the reduced curvature </b><b><i>r_sheetM</i></b><b>₁.</b> The results are calculated for vanishing preferred or spontaneous curvatures <i>m</i>₁ = <i>m</i>₂ = <i>m</i>₃ = 0 and vanishing curvature asymmetry <i>m</i>₁₂ = 0; see Equation 8 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032753#pone.0032753.s001" target="_blank">Text S1</a> for the definition of . The reduced curvature <i>r_sheetM</i>₁ of the cup shapes can be positive or negative, which distinguishes between upward and downward bending of the sheet as schematically illustrated in the top row of the figure. For <i>r_sheet</i>/<i>r_rim</i><5.1 the sheet represents the shape of minimal energy. At <i>r_sheet</i>/<i>r_rim</i> = 5.1 the flat sheet and the closed double-membrane vesicle are local minima with the same energy, but separated by a considerable energy barrier preventing the shape transition. Increasing the effective size of the vesicle decreases the barrier continuously. At the critical size, <i>r_sheet</i>/<i>r_rim</i> = 10.2, the energy barrier disappears and the sheet becomes unstable with respect to arbitrarily small perturbations, which transforms the sheet into a closed vesicle. Energy landscapes of asymmetric sheets with nonzero curvature asymmetry <i>m</i>₁₂ are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032753#pone.0032753.s005" target="_blank">Fig. S3</a>.</p

Stability diagram of double-membrane sheets as a function of preferred or spontaneous rim curvature <i>m</i>₃ and sheet size <i>r_sheet</i>.

<p>Both the rim curvature and sheet size are given in units of the rim curvature radius <i>r_rim</i>. The sheets are (almost) symmetric in the sense that their two faces have similar preferred curvatures, <i>m</i>₁≅<i>m</i>₂ and the curvature asymmetry <i>m</i>₁₂ is small compared to 1/<i>r_rim</i>. The regime of stable sheets (gray area) is bounded by an instability line corresponding to the critical sheet size as described by Equation 10 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032753#pone.0032753.s001" target="_blank">Text S1</a>. The instability line has two branches for <i>m₃r_rim</i><1/2 and <i>m₃r_rim</i>>1/2. For nonzero <i>m</i>₁₂ the two branches meet at the maximal critical disk size as given by 2/<i>|m₁₂|</i>. The latter size diverges for vanishing curvature asymmetry <i>m₁₂</i> = 0, i.e., in this case, an arbitrarily large sheet remains stable. If the sheet reaches the instability line by lateral growth, protein adsorption and/or desorption at its rim (long arrows), it closes into a double-membrane vesicle. Sheets above the instability line are unstable and close into such vesicles as well. The broken horizontal line with <i>r_sheet</i>/<i>r_rim</i>≅45 corresponds to the autophagosome in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032753#pone-0032753-g002" target="_blank">Fig. 2C</a> with diameter <i>r</i>⁰<i>_sheet</i>≅900 nm and <i>r_rim</i>≅20 nm. The intersection of the broken line with the two branches of the instability line determines the preferred or spontaneous rim curvature <i>m</i>₃≅1/(76 nm) or 1/(28 nm) (arrowheads) of the unstable sheet that preceded the autophagosome in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032753#pone-0032753-g002" target="_blank">Fig. 2C</a>.</p

Membrane Morphology Is Actively Transformed by Covalent Binding of the Protein Atg8 to PE-Lipids

<div><p>Autophagy is a cellular degradation pathway involving the shape transformation of lipid bilayers. During the onset of autophagy, the water-soluble protein Atg8 binds covalently to phosphatdylethanolamines (PEs) in the membrane in an ubiquitin-like reaction coupled to ATP hydrolysis. We reconstituted the Atg8 conjugation system in giant and nm-sized vesicles with a minimal set of enzymes and observed that formation of Atg8-PE on giant vesicles can cause substantial tubulation of membranes even in the absence of Atg12-Atg5-Atg16. Our findings show that ubiquitin-like processes can actively change properties of lipid membranes and that membrane crowding by proteins can be dynamically regulated in cells. Furthermore we provide evidence for curvature sorting of Atg8-PE. Curvature generation and sorting are directly linked to organelle shapes and, thus, to biological function. Our results suggest that a positive feedback exists between the ubiquitin-like reaction and the membrane curvature, which is important for dynamic shape changes of cell membranes, such as those involved in the formation of autophagosomes.</p></div

Atg8-PE stabilizes membrane curvature and sorts into strongly curved membanes.

<p>(<b>A</b>) Setup used for tube pulling. After aspiration, the vesicle containing biotinolyted lipids and the streptavidin coated bead were brought in contact and adhere. Once bound, a tube was pulled by moving the pipette holding the bead, a confocal xy-scan illustrates such a membrane tube pulled from a GUV. (<b>B</b>) Fluorescence intensity profiles of the membrane tube (top) and the corresponding GUV membrane (bottom) of the boxed regions in (<b>A</b>). (<b>C</b>) At high Atg8-PE densities, pulled tubes appeared blurry and exhibit pronounced fluctuations. Two consecutive xy-scans of the same tube illustrate movements of the tube; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115357#pone.0115357.s001" target="_blank">S1 Video</a>. (<b>D</b>) Increasing the membrane tension of the GUV at time t = 1 min (blue line, right axis) decreases the radius of the membrane tube from 70 nm to 16 nm and accordingly changes the intensities of the membrane tube and Atg8-PE (data points, left axis). This change is reversible as shown with decreasing the tension at time t = 7 min. For experimental details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115357#s4" target="_blank">methods</a>. Data points correspond to consecutive scans of one individual tube. (<b>E</b>) The normalized Atg8-PE densities in the tube D^tube/D^GUV increase with tension. Three different Atg8 concentrations are shown, n = 7 GUVs for 0.56 and 0.63 µM Atg8, n = 3 GUVs for 0.75 µM Atg8, mean ± sem. The scale bars in (<b>A</b>) and (<b>C</b>) are 5 µm.</p

Minimal system to covalently bind the protein Atg8 via an ubiquitin-like reaction to phosphatidylethanolamine (PE).

<p>Minimal system to covalently bind the protein Atg8 via an ubiquitin-like reaction to phosphatidylethanolamine (PE).</p

Atg8-PE density on GUVs depends on Atg8 concentration.

<p>(<b>A</b>) Atg8-PE densities on isolated GUVs (D^GUV) and in adhesion zones between two GUVs (D^AZ) as a function of the bulk Atg8 concentration; insets show adhering GUVs for the indicated concentrations; mean ± SEM, n = 9–12 GUVs per concentration. At the highest Atg8-PE density examined, GUVs spontaneously form external protrusions (<b>B</b>), while some GUVs disintegrate into tubular networks (<b>C, D</b>) or show pronounced non-fluctuating deformations (<b>E, F</b>). This behavior is not observed in the absence of ATP (<b>G</b>). All snapshots are merged dual color images; scale bars 10 µm.</p

Curvature preference of the Atg8 conjugation reaction.

<p>(<b>A</b>) Conjugation of Atg8 to individual liposomes of varying size as measured by the SLiC-assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115357#pone.0115357-Hatzakis1" target="_blank">[25]</a>. The Atg8-PE density, , is plotted as a function of the vesicle radius, , after 20 min incubation with 125 nM Atg8 for n = 366 vesicles. The insets show snapshots of immobilized liposomes. (<b>B</b>) Atg8-PE density (same data) as a function of membrane curvature, .</p