Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir - PubMed (original) (raw)

Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir

Sylvia Neumann et al. Nat Protoc. 2013 Jan.

Abstract

A complete understanding of the molecular mechanisms governing vesicle formation requires quantitative assays and vesicle reconstitution using purified components. We describe a simple model membrane template for studying protein-mediated membrane remodeling and vesicle formation or fission that is amenable to both quantitative biochemical analysis and real-time imaging by epifluorescence microscopy. Supported bilayers with excess membrane reservoir (SUPER) templates are compositionally well-defined unilamellar membrane systems prepared on 2-5-μm silica beads under conditions that enable incorporation of excess membrane to form a loosely fitting bilayer that can be used to study membrane remodeling and fission. This protocol describes methods for SUPER template formation and characterization, as well as for the qualitative observation and quantitative measurement of vesicle formation and fission via microscopy and a simple sedimentation assay. SUPER templates can be prepared within 60 min. Results from either sedimentation-based or microscopy-based assays can be obtained within an additional 60 min.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

Figures

Figure 1

Figure 1

Generation of SUPER templates. (a) To generate SUPER templates, fluorescently labeled liposomes that contain negatively charged lipids are deposited onto 5-μm silica beads under high-salt conditions, so as to incorporate excess membrane reservoir. (b) The incorporation of membrane reservoir can be visualized by adding the templates onto a clean, uncoated glass coverslip in a droplet of assay buffer. The templates will bind to the glass surface, and the excess membrane will spill out to form a supported bilayer surrounding the silica bead (Supplementary Video 1). Scale bar, 5 μm.

Figure 2

Figure 2

Sedimentation-based assay. (a) To quantify vesicle release, SUPER templates are incubated with dynamin-1 in the presence of nucleotides. Templates can then be easily pelleted at low speed owing to their size, and any released vesicles will remain in the supernatant. The total incorporation of membrane is determined by Triton X-100 extraction of the templates. (b) Carefully add SUPER templates to the top of the reaction mixture for a sedimentation assay without performing any additional mixing (Supplementary Video 2). RT, room temperature.

Figure 3

Figure 3

Real-time visualization of membrane fission. (a) SUPER templates are applied to a BSA-coated Lab-Tek chamber or coverslip for microscopy-based assays. Dynamin is added from a pipette to initiate the reaction. (b) SUPER templates are added to a well of a Lab-Tek chamber. (c) Spread SUPER templates evenly throughout the well by slowly moving the pipette according to the schematic while emptying the tip. (d) Once the templates are settled, transfer the well onto a microscope stage, begin to record a video and carefully add dynamin to the well.

Figure 4

Figure 4

Sedimentation and microscopy assays for vesicle release. (a) Representative results from a sedimentation fission assay. SUPER templates were incubated in 20 mM HEPES, 150 mM KCl and 1 mM MgCl2 for 30 min in the presence of 1 mM GTP and with increasing amounts of dynamin (Dyn)-1. Vesicle release is dependent on dynamin-1 presence and concentration and requires GTP. (b) Fluorescence micrograph of released vesicles. (c) SUPER templates were dispersed into assay buffer containing an oxygen-scavenging system and 1 mM GTP in individual wells of an eight-well glass slide. After the beads had settled, imaging was started and Dyn-1 was added to the solution with a pipette to a final concentration of 0.5 μM. The figure shows the field of view before (left) and after (right) addition of Dyn-1. See Supplementary Video 3 for the full sequence. Scale bars, 5 μm.

Figure 5

Figure 5

Fission of membrane tethers. (a) SUPER templates are applied to a BSA-coated Lab-Tek chamber. After the templates settle, untreated silica beads of sizes (diameter = 20 μm) bigger than those used to produce the SUPER templates are added to the microscopy chamber in order to create tethers. Owing to their size, the 20-μm beads readily settle within the pipette tip. (b) Only the volume of the mixture that contains the settled beads is added into one corner of the well. (c) When they are first added to the wells and rolled over the templates, the larger beads clump. (d) Gently roll the clumped larger beads over the templates another three or four times by tilting the chamber in a W-shaped path. (Supplementary Video 4).

Figure 6

Figure 6

Time course of membrane tether fission. Tethers were drawn from SUPER templates as described in Figure 5 and text. After imaging was started, dynamin-1 was added to the solution (t = 0) with a pipette to a final concentration of 0.5 μM. See Supplementary Video 5 for the full sequence. Scale bar, 5 μm.

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