Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.
Mitochondrial dynamics is essential for the organelle’s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.
Membrane compartmentalization is a hallmark of eukaryotic cells1 (Figure 1A). Biological membranes are increasingly recognized as more than a two-dimensional solvent, and are considered as an environment playing critical roles in regulating protein function and macromolecular complex assembly2,3. Native lipids are ligands that regulate membrane protein activity3,4. Membrane spatial organization and the ability of membranes to be sculpted into diverse shapes are important physical properties for selecting new functions3,5.
Model membrane platforms are biomimetic systems that can help us understand cellular membrane structure, dynamics, and function6,7,8. Model membranes typically comprise a lipid mixture of well-defined composition, with defined biophysical properties (stiffness, thickness, and elasticity). Coupled to fluorescence imaging, model membrane platforms allow quantitative analysis of membrane structure and function9,10,11. Lipid bilayer reconstitution strategies have been used to study SNARE-mediated membrane fusion9,10, DNA-mediated membrane fusion12, and viral fusion11,13. An advantage of such methods is the potential to obtain kinetic information for intermediate steps preceding an observable reaction event14.
The plasma membrane has been extensively studied using model membranes. Bilayers with lipid phase separation have been developed to study lipid raft structures important in cellular signaling11,15,16. Micropatterned lipid planar bilayers17,18 have been used to investigate the organization of cell receptors. Polymer or gel-supported membranes have been used as biomimetic systems to study the membrane-cytoskeleton organization, membrane protein partitioning during cell signaling, and migration at cell-cell contacts19.
Artificial membrane systems are also being applied to study subcellular organelles20. Organelles feature characteristic morphologies that create distinct sub-environments. The endoplasmic reticulum (ER) network is one example. Upon reconstitution of reticulons into liposomes, tubular membrane structures with properties similar to the cellular ER are formed21. The addition of atlastin, an ER fusion protein, can induce lipid tubules from liposomes to form a network20. This is one example for how proteoliposomes can provide functional insight into organelle morphology and dynamics.
Mitochondrial membrane fusion and fission are essential for the health of the mitochondrial population22,23,24,25. A set of dynamin family GTPases catalyzes mitochondria membrane fusion. Mfn 1/2 catalyzes outer-membrane fusion. Opa1 mediates inner-membrane fusion26 (Figure 1B). Opa1 has two forms: a long form (l-Opa1), transmembrane-anchored to the mitochondrial inner-membrane, and a ‘soluble’ short form (s-Opa1), present in the intermembrane space. The ratio of the two Opa1 forms is regulated by the activity of two proteases, Oma1 and Yme1L27,28,29,30. Important questions in Opa1 regulation include: how the two forms of Opa1, (short and long) mediate membrane fusion and their regulatory interplay28,29,31,32,33.
Here we describe a reconstitution strategy successfully applied to investigate mitochondrial inner-membrane fusion that clarified the roles of l- and s-Opa1 in inner-membrane fusion. We developed a platform mimicking the mitochondrial inner-membrane using a polymer-tethered lipid bilayer and 200 nm unilamellar vesicles. The benefits of a polymer tether beneath the lipid bilayer include the following. First, it preserves the reconstituted transmembrane protein, which would otherwise may be disrupted by the proximity to the glass slide34. Secondly, it serves a thick water layer between the lipid bilayer and glass substrate, which facilitates studies of pore opening9, and thirdly the viscoelastic nature of the PEG polymer allows membrane curvature changes35. We used three-color fluorescence imaging to characterize steps in membrane fusion (Figure 1C-F).
Figure 1: Monitoring mitochondrial membrane fusion.
(A) Organelles are cellular membrane compartments. (B) Sequential steps of mitochondrial membrane fusion. Fusion of the outer membrane of mitochondria is catalyzed by Mfn1 and/or Mfn2, while inner-membrane fusion is mediated by Opa1. (C-F) Schematic of the in vitro reconstitution platform to study mitochondrial membrane fusion. The platform includes two parts: a proteoliposome and a polymer-tethered lipid bilayer, both with reconstituted l-Opa1. Fluorescent labels, including two different fluorescent membrane dyes and a content marker, help distinguish steps during membrane fusion. The two membrane markers (Cy5-PE (red) and TexasRed PE (orange) make a FRET pair, which can report on close membrane docking. Diffusion of TexasRed-PE that labels proteoliposome is an indicator of lipid demixing (hemifusion). Content release is monitored through the dequenching of the calcein signal (shown in green). Panels A and B created using Biorender. Please click here to view a larger version of this figure.
1. Preparation of lipid mixtures
2. Fabrication of lipid bilayers
Figure 2: Steps in making a polymer-tethered lipid bilayer.
Steps of making lipid bilayers using Langmuir-Blodgett dipping (A-C) and Langmuir-Schaefer transfer (D) techniques. (E) The final “sandwich” containing the lipid bilayer. Please click here to view a larger version of this figure.
3. Protein reconstitution into the polymer-tethered lipid bilayer
Figure 3: Procedure for reconstituting l-Opa1 into a polymer-tethered lipid bilayer. Please click here to view a larger version of this figure.
4. Preparation of proteoliposomes
5. Imaging and data analysis
The reconstituted transmembrane protein freely diffuses and is homogeneously distributed in the membrane.
Example images of a lipid bilayer and its lipid fluidity validated by epifluorescence microscopy is shown in Figure 4. Lipid distribution in bilayer before and after photobleaching is shown in Figure 4A,B. Homogeneity of the lipid bilayer was visualized using an epifluorescence microscope before and after reconstitution (Figure 4D,E). l-Opa1 reconstituted in lipid bilayer was validated by fluorescence correlation spectroscopy (FCS). We use dye conjugated lipids to evaluate the lipid diffusivity of the bilayer. Reconstituted Opa1 was labeled using a fluorescent-tagged anti-Opa1 C-terminal antibody. Bilayer lipid diffusion was measured as 1.46 ± 0.12 μm2/s, while the diffusion coefficient of bilayer-reconstituted l-Opa1 was 0.88 ± 0.10 μm2/s. Intensity readout from the FCS curves indicated 75% of l-Opa1 is reconstituted into the lipid bilayer (Figure 4G,H). These results suggest that l-Opa1 freely diffuses in the polymer-tethered lipid bilayer with the potential to self-assemble into functional complexes.
Figure 4: Distribution of lipid and reconstituted protein in the model membrane.
(A-C) Example images of a lipid bilayer and its lipid fluidity validated by epifluorescence microscopy. (A) Homogeneous lipid distribution in bilayer prior to photobleaching. (B) Snapshot immediately after photobleaching. (C) Bilayer imaged after fluorescence recovery indicates good lipid fluidity of the membrane following reconstitution. (D,E) Representative images of lipid distribution before (D), and after (E) l-Opa1 reconstitution indicate the reconstitution process did not create defects in the bilayer. Representative TIRF image of l-Opa1 labeled with Alexa 488 conjugated antibody (F) showing a homogeneous distribution of Opa1 upon reconstitution. G. Representative raw photon counts of l-Opa1 signal by fluorescent correlation spectroscopy. In the control, no l-Opa1 was reconstituted in the bilayer, while antibody was added and rinsed. The diffusion of l-Opa1 is significantly slower than lipids in the membrane, consistent with successful reconstitution of transmembrane l-Opa1 (H). Scale bar: 10 µm. Please click here to view a larger version of this figure.
Fluorescence step bleaching indicated an average of 2-3 copies of l-Opa1 were reconstituted in a given liposome (Figure 5A,B). The size distribution of Opa1 reconstituted proteoliposomes was tested after reconstitution using DLS (Figure 5C). The reconstitution of Opa1 in proteoliposomes was also verified using FCS. The diffusion coefficient of free antibody was 164 ± 22 μm2/s; diffusion coefficient for liposomes labeled with a lipid dye was 2.22 ± 0.33 μm2/s, and the diffusion coefficient for l-Opa1 proteoliposomes bound to a TexasRed labeled anti-His antibody was 2.12 ± 0.36 μm2/s.
Figure 5: Fabrication and characterization of proteoliposomes.
(A) Steps in fabricating proteoliposomes with encapsulated, quenched calcein. (B) Representative data of fluorescent step-bleaching show an average of 2-3 copies of l-Opa1 embedded in the liposome. (C) Representative size distributions of proteoliposomes (red) without any nucleotide 1 h after GTP incubation (green). Please click here to view a larger version of this figure.
Detection of membrane tethering, lipid demixing/hemifusion, and pore opening by fluorescent microscopy.
Membrane tethering is monitored by observing the signal of TexasRed on the surface of lipid bilayer using TIRF microscopy (Figure 6A). Membrane lipid demixing (hemifusion) behavior was monitored through TexasRed as the dye diffuses from the liposomes into the lipid bilayer. Calcein dequenching helps distinguish full fusion pore formation from only lipid demixing. This allows comparison between conditions where particles stall at hemifusion (Fig 6B), and particles that proceed to full fusion (Figure 6C).
Membrane tethering is indicated by a stable lipid signal from liposomes. The distance could be evaluated based on the FRET signal between the labels of the two membranes36. Hemifusion signal features no dequenching in the calcein signal (Figure 6B, lower row), but a rapid decay of the TexasRed signal indicates diffusion of the dye into the lipid bilayer (Figure 6B upper row). Full fusion (with pore opening) features both lipid decay and content release (Figure 6C). TexasRed intensity and calcein intensity can be tracked in a time-dependent manner to provide quantitative detail for the kinetics of membrane fusion36.
Figure 6: Representative results showing particle tethering (A, scale bar 10 µm), hemifusion (B, scale bar 0.5 µm), and fusion (C, scale bar 0.5 µm).
(A) Proteoliposomes tethered to Opa1-reconstituted lipid bilayer before GTP addition. (B) An example of hemifusion. The upper row in B shows lipid demixing (TexasRed signal, red), lower row in B shows no content release (calcein signal, green) under these conditions. (C) A representative trace of proteoliposome fusing with the lipid bilayer. Content release can be observed from images in the lower row of showing dequenching of calcein (lower row, green). Please click here to view a larger version of this figure.
In vitro model-membrane systems can describe complex membrane processes under well-defined conditions. These systems can distinguish minimal components necessary for complex molecular processes to reveal molecular mechanisms6,15,20,38. For membrane proteins, liposomes and planar supported bilayers are common reconstitution systems. In contrast to solid-supported lipid bilayers, the polymer cushion between the substrate and supported membrane in polymer-tethered bilayers allows free mobility of large membrane proteins, and transmembrane-proteins to diffuse and assemble freely34. These features helped us investigate the kinetics of mitochondria inner-membrane fusion36.
We prepared a polymer-tethered lipid bilayer using Langmuir-Blodgett/Langmuir-Schaefer (LB/LS) techniques. This allows us to prepare a bilayer with asymmetric lipid components. Cellular membranes have asymmetric leaflet composition, and the LB/LS approach allows the study of such bilayers. With Schaefer transfer, an entire glass substrate can be covered by a lipid bilayer. It is critical to prepare a clean surface for bilayer preparation. Additionally, it takes practice to perform a Schaefer transfer correctly. Unsuccessful Schaefer transfer can create unwanted defects in a lipid bilayer. In this protocol, the pressure added to the film balance is applicable for a bilayer containing 20% cardiolipin. For bilayers with other components, refer to the surface pressure-area isotherm of the key components. An alternative method is the Langmuir-Blodgett/vesicle fusion (LB/VF) method, where the bottom lipid monolayer is transferred from the air-water interface of a Langmuir trough onto a clean substrate, then liposomes fuse to the top of the supported lipid monolayer and form the final bilayer39. Reconstitution of membrane proteins using the LB/VF method is more straightforward than LB/LS, as reconstitution can be performed through the fusion of proteoliposomes. However, vesicle fusion requires the addition of excess liposomes, which may complicate the study of membrane events dependent on concentration-dependent protein-protein interactions.
The successful reconstitution of transmembrane proteins into both polymer-tethered lipid bilayers and liposomes in a preferred functional orientation is important, yet difficult to enforce. Experimental controls are needed to account for this. For polymer-tethered lipid bilayers, it is also important to maintain the integrity of lipid bilayer during reconstitution. Surfactant concentrations must be kept relatively low to prevent dissolving the lipid bilayer, but high enough to prevent denaturation of the protein of interest37,40. The method described here is ideal for reconstituting membrane proteins for single-molecule studies but is not necessarily scalable for larger-scale studies. Surfactant choice is another important consideration. Frequently, the surfactant used for purification and storage is a good starting point. The maximum concentration of surfactant is usually ~200 times less of the CMC36, in a range where the surfactant maintains protein stability and prevents protein aggregation, while maintaining the integrity of membrane36. Cocktails containing 2 or 3 surfactants may be considered. For reconstitution into liposomes, a low concentration of surfactant is not necessary. However, surfactant concentrations below CMC are preferable to maintain uniform size and morphology distribution for the liposomes. To prevent leakage of content dye, it is necessary to dialyze against a dye-containing buffer.
In contrast to liposome-based fusion assays, the platform we established provides an approach to investigate the kinetics of each step of membrane fusion. This method provides the ability to study transmembrane fusion proteins under near-native conditions. Model membrane platforms can be applied to study membrane protein assembly and oligomerization, membrane “sculpting”, and protein-lipid interactions of proteins in subcellular environments, like the mitochondrial inner-membrane. This method also allows exploration of important physiological conditions in the membrane-protein interplay, such as bilayer composition asymmetry. The role of a key mitochondrial lipid, cardiolipin, in the bilayer properties of both liposomes and polymer-supported bilayers remains to be defined. Properties such as the ionic strength, membrane thickness, membrane stiffness, membrane curvature, and membrane elastic-viscosity properties all may influence the ability of proteins to assembly into specific functional states. Future studies creatively applying model membrane systems have potential to uncover new aspects of membrane protein organization and function.
The authors have nothing to disclose.
The authors acknowledge the support from Charles H. Hood Foundation Child Health Research Award and generous support from the Department of Molecular Biology at Massachusetts General Hospital.
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5) | Avanti polar lipid | Cat #: 810335C1mg | membrane fluorescent markers |
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) | Avanti Polar lipids | Cat #: 880130P | lipid molecules |
1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (sodium salt) | Avanti Polar lipids | Cat #: 710335P | lipid molecules |
18:1 (Δ9-Cis) PC (DOPC) | Avanti Polar lipids | Cat #: 850375P | lipid molecules |
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine | Avanti Polar lipids | Cat #: 850757P | lipid molecules |
Alexa Fluor 488 Antibody Labeling Kit | ThermoFisher Scientific | A20181 | |
Amber vial with PTFE liner | Fisher scientific | 14-955-332 | sample vials to keep lipid solutions |
Calcein | Sigma-Aldrich | Cat #: C0875; PubChem Substance ID: 24892279 | fluorescent dye |
Chloroform | Fisher scientific | 298-500/ C295-4 | Fisher brand Chloroform is usually quite reliable for lipid works. |
Concavity slide (1 well) | Electron Microscopy Science | 71878-05 | applied as Schaefer Slide |
FCS analysis tool | Smith Lab, University of Akron | software tool | |
Fiji /ImageJ | Fiji | SCR_002285 | software tool |
Fisherbrand Cover Glasses: Circles | Fisher scientific | 12-545-102 | Cover glass for solid supported lipid bilayers, the item is now discontinued as authors prepared the manuscript. An alternative is Fisher brand premium cover glass with catalog number: 12-548-5M |
GTP Disodium salt | SIGMA-ALDRICH INC | Cat #: 10106399001 | |
Langmuir & Langmuir-Blodgett Trough | Biolin Scientifc | KN2002 | |
L-α-lysophosphatidylinositol (Liver, Bovine) (sodium salt) | Avanti Polar lipids | Cat #: 850091P | lipid molecules |
Mini Extruder | Avanti Polar lipids | 610020 | |
n-Dodecyl-β-D-Maltopyranoside | Anatrace | Cat #: D310 25 GM | surfactant for reconstitution |
n-Octyl-α-D-Glucopyranoside | Anatrace | Cat #: O311HA 25 GM | surfactant for reconstitution |
PC Membranes 0.2μm | Avanti Polar Lipids | 610006 | |
Rabbit Anti-Opa1 antibody | NOVUS BIOLOGICALS | Cat #: NBP2-59770 | antibody for Opa1 C-terminal detection |
Slidebook | Intelligent imaging | RRID: SCR_014300 | software tool |
Teflon threaded seal tape | Fisher Scientific | NC0636085 | taflon tape for sample storage |
Texas Red 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Triethylammonium Salt (Texas Red DHPE) | ThermoFisher Scientific | Cat #: T1395MP | membrane fluorescent markers |