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. 2017 Jan 15;28(2):309-321.
doi: 10.1091/mbc.E15-11-0759. Epub 2016 Nov 23.

How and why intralumenal membrane fragments form during vacuolar lysosome fusion

Sevan Mattie  1 , Erin K McNally  1 , Mahmoud A Karim  1 , Hojatollah Vali  2 , Christopher L Brett  3   2
Affiliations

How and why intralumenal membrane fragments form during vacuolar lysosome fusion

Sevan Mattie et al. Mol Biol Cell. .

Abstract

Lysosomal membrane fusion mediates the last step of the autophagy and endocytosis pathways and supports organelle remodeling and biogenesis. Because fusogenic proteins and lipids concentrate in a ring at the vertex between apposing organelle membranes, the encircled area of membrane can be severed and internalized within the lumen as a fragment upon lipid bilayer fusion. How or why this intralumenal fragment forms during fusion, however, is not entirely clear. To better understand this process, we studied fragment formation during homotypic vacuolar lysosome membrane fusion in Saccharomyces cerevisiae Using cell-free fusion assays and light microscopy, we find that GTPase activation and trans-SNARE complex zippering have opposing effects on fragment formation and verify that this affects the morphology of the fusion product and regulates transporter protein degradation. We show that fragment formwation is limited by stalk expansion, a key intermediate of the lipid bilayer fusion reaction. Using electron microscopy, we present images of hemifusion diaphragms that form as stalks expand and propose a model describing how the fusion machinery regulates fragment formation during lysosome fusion to control morphology and protein lifetimes.

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Figures

FIGURE 1:
FIGURE 1:
Fragment formation during vacuolar lysosome fusion. (A) Working model describing how intralumenal membrane fragments form during homotypic vacuolar lysosome membrane fusion. Numbers indicate reaction intermediates that were visualized using TEM and are shown in Figure 5D. (B) Live yeast cells stained with FM4-64 to label vacuole membranes were imaged using HILO microscopy. Examples of vacuole fusion events are shown as a series of inverted micrographic images acquired over time (minutes). Dashed lines outline each cell observed by differential interference contrast. Scale bars, 1 µm.
FIGURE 2:
FIGURE 2:
GTPγS and rVam7 have opposing effects on the time interval between stalk and pore formation, fragment formation, and surface area during vacuole fusion. Lipid mixing (A) or content mixing (B) was recorded during in vitro homotypic vacuole fusion reactions in the absence (control) or presence of 100 nM rVam7, 0.2 mM GTPγS, or both (n ≥ 6). As negative control, 3.2 µM Gyp1-46, a Rab-GTPase inhibitor, was added to reactions to prevent fusion. (C) Maximum lipid mixing and content mixing values observed as ratios for each condition. (D) Times at half-maximal lipid mixing (circles) and content mixing (squares); time intervals between events were calculated (numbers) and are shown (arrow). (E) Images of FM4-64 stained vacuoles obtained 60 min after fusion was initiated. Arrowheads indicate intralumenal fragments. Scale bar, 2 µm. Using micrographs of fusion reactions, we calculated the percentage of vacuoles with intralumenal membrane fragments (F) and compared these values to time intervals between stalk and pore formation (G), boundary length (H), or the change in surface area relative to number of fusion events (as assessed by content mixing; I, J) for each condition (n = 324–802). Asterisks indicate data points significantly different from control (p < 0.05).
FIGURE 3:
FIGURE 3:
Hemifusion diaphragm expansion inversely correlates with fragment formation. (A) Reasoning behind the assay used to detect hemifusion diaphragms by HILO microscopy during homotypic vacuole fusion. Fluorescence micrographs of vacuoles isolated from yeast expressing Vph1-GFP (B) or Cot1-GFP (C) and stained with FM4-64 acquired 30 min after fusion were initiated in the presence of 0.2 mM GTPγS, 100 nM rVam7, or both. Interfaces containing (closed arrowheads) or lacking (open arrowheads) GFP fluorescence are indicated. Left, GFP fluorescence intensity profile plots. Scale bars, 1 µm. The percentage of vacuole contact sites that do not contain Vph1-GFP or Cot1-GFP (D) was calculated and compared with the percentage of vacuoles with internal fragments (E, F; n ≥ 132). Asterisks indicate data points significantly different from control (p < 0.05).
FIGURE 4:
FIGURE 4:
Vacuole membrane docking and hemifusion visualized by transmission electron microscopy. (A) Transmission electron micrograph of a vacuole fusion reaction under control conditions (+ATP) at 30 min. Boxes are higher-magnification images illustrating a docking site between apposing organelles. Scale bars, 200 nm. Images of vacuoles incubated without ATP (B) or with ATP and recombinant Gdi1 protein, a Rab-GTPase inhibitor (14 µm; C) are shown as negative controls. Scale bars, 200 nm. (D) Top, transmission electron micrographs of docked vacuole membranes, a hemifusion diaphragm, and ruptured diaphragm. Bottom, higher-magnification images of membrane interfaces. Scale bars, 500 nm. (E) Averages of linear density plots within the areas shown in D (n = 10–88; error bars represent SD). Scale bars, 50 nm.
FIGURE 5:
FIGURE 5:
Vacuole membrane hemifusion confirmed by electron tomography and cryo–electron microscopy. (A) Top left, vacuole hemifusion diaphragm obtained using electron tomography. Serial sections of the boxed area containing the interface are shown (right; Supplemental Video S3). Scale bar, 500 nm. Bottom left, three-dimensional reconstruction of the interface at higher magnification. Supplemental Video S4 illustrates the reconstruction. (B) Top, cryo–electron micrograph of an in vitro vacuole fusion reaction. Examples of docked vacuole membranes (middle) and a hemifusion diaphragm (bottom) are shown at high magnification. Scale bars, 50 nm.
FIGURE 6:
FIGURE 6:
Characterization of hemifusion intermediates that underlie fragment formation and their potential contribution to cellular physiology. (A) Top, transmission electron micrographs of vacuole fusion reactions in the absence and presence of 0.2 mM GTPγS obtained at 30 min or 100 nM rVam7 at 10 min. Bottom, interfaces at higher magnification. Scale bars, 250 nm. Electron micrographs were used to calculate (B) percentages of vacuoles engaged in docking or hemifusion (n ≥ 200) or (C) intermembrane distances at docked interfaces (n ≥ 13). (D) Transmission electron micrographs showing examples of different hemifusion intermediates observed in vacuole fusion reactions containing 0.2 mM GTPγS. Left, higher-resolution images of interfaces between adjacent vacuoles shown on the right. Numbers correspond to intermediates of the vacuole membrane fusion reaction illustrated in Figure 1A. Red arrowheads indicate hemifusion diaphragms. Scale bars, 500 nm. (E) Model describing how intralumenal fragment formation is regulated and may contribute to changes in lysosomal vacuole physiology in response to cellular cues.
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