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Aug 2018

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Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion
巨型囊泡融合在负曲面膜上的顺序蛋白重建及动态   

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Abstract

In vitro investigation of the interaction between proteins and positively curved membranes can be performed using a classic nanotube pulling method. However, characterizing protein interaction with negatively curved membranes still represents a formidable challenge. Here, we describe our recently developed approach based on laser-triggered Giant Unilamellar Vesicles (GUVs) fusion. Our protocol allows sequential addition of proteins to a negatively curved membrane, while at the same time controlling the buffer composition, lipid composition and membrane tension. Moreover, this method does not require a step of protein detachment, greatly simplifying the process of protein encapsulation over existing methods.

Keywords: GUV (GUV), Membrane fusion (膜融合), Negatively curved membrane (负弯曲的膜), ESCRT (ESCRT), CHMP (CHMP), Bottom-up (自下而上), Protein encapsulation (蛋白质包封), Nanotube pulling (纳米管牵引)

Background

Cellular membrane remodeling processes including vesicle trafficking can be topologically split into either outside-in budding such as endocytosis and inside-out budding such as intraluminal vesicle formation at early endosomes, enveloped virus budding and cytokinesis. A budding event induces changes in membrane shape, resulting in two distinct topologies: a convex surface or a concave surface, conventionally defined as positive and negative curvature, respectively (Figure 1).

In vitro methods for studying outside-in budding processes based on membrane nanotube pulling such as the interaction of proteins with positively curved membranes during endocytosis are well-established (Sorre et al., 2012; Prévost et al., 2017). In contrast, investigating protein interaction with negatively curved membranes is much more challenging because it is in general not straightforward to obtain such a topology–i.e., to incorporate proteins inside a membrane nanotube pulled from a GUV in a classic tube-pulling experiment. Indeed, the membrane has to be deformed away from the compartment where proteins are present (Figure 1). Encapsulating the protein of interest inside the GUV can in principle be achieved during GUV swelling, and by pulling out a nanotube afterwards (Prévost et al., 2015). This methodology, however, requires the protein to be detached from the Outer leaflet of the GUV, which can only be performed if the binding is reversible, and the procedure has to be adjusted for each individual protein. Moreover, when a protein is encapsulated, it also interacts with the membrane during swelling, potentially hampering the process of swelling itself. Another limitation of this protocol is that sequential addition of proteins (or lipids) is not possible.

An alternative method consists in pulling a nanotube inside the GUV, and adding the protein from the outside by micropipette injection (Dasgupta and Dimova, 2014). But this method requires very flaccid GUVs, a combination of flow and optical tweezers to form the tube, and is not suitable when the tube diameter has to be controlled down to 15-20 nm.

Here we describe a protocol to investigate the interaction of proteins with negatively curved membranes and membrane necks (De Franceschi et al., 2018). We apply this method to study the endosomal sorting complex required for transport (ESCRT) machinery, which catalyzes inside-out membrane budding processes by polymerizing on negatively curved membranes that carry a negative charge (Henne et al., 2013; McCullough et al., 2018; Caillat et al., 2019). Characterizing the ESCRT-III complex is particularly challenging, because these proteins exhibit strong binding to the membrane, so that their removal from the Outer membrane of a GUV after the encapsulation process is impossible (De Franceschi et al., 2018), making other methods (Prévost et al., 2015) unsuitable. Our method relies on laser-triggered GUV fusion (Rørvig-Lund et al., 2015), and presents a number of advantages over existing methods. First, protein encapsulation is performed in conditions that inhibit protein binding to the membrane, avoiding inhibition of GUV swelling and protein/lipids aggregation. Thus, the protein can be encapsulated using a buffer that keeps it stable and avoids unwanted in-bulk polymerization. However, upon fusion with a protein-free GUV, optimal binding conditions, including lipid species and salt concentration, can be achieved. Importantly, GUV fusion is triggered after a membrane nanotube has been pulled, therefore allowing visualizing dynamic interaction of proteins with a negatively-curved membrane topology. Moreover, by performing multiple fusion events, additional proteins can be added sequentially. Similarly, the lipid composition can be modulated in a controlled fashion by sequential fusion events.


Figure 1. Schematic representing outside-in and inside-out budding events, and the membrane curvatures created during these processes

Method outline: The principle in this methodology is to have proteins inside one neutral GUV (green in Figure 2) and a second protein-free GUV made of negatively charged lipids (magenta in Figure 2) until GUV fusion is triggered. A tube is pulled beforehand from the negatively charged GUV (magenta), so that upon GUV fusion negative membrane curvature becomes accessible to the proteins. Therefore, the lipid mix and buffer in the green GUV are designed to maintain proteins' stability and allow their encapsulation avoiding premature binding and aggregation. At the same time, the final lipid and buffer composition resulting from GUV fusion must create the appropriate conditions for protein-membrane interaction.

We exemplify this concept by explaining the design of buffers and lipid mixes used for investigating binding between the ESCRT-III protein CHMP2B and PI(4,5)P2 (De Franceschi et al., 2018).

CHMP2B is stable at relatively high salt concentration. Lowering NaCl concentration to 50 mM in the presence of negatively charged lipids such as PI(4,5)P2 triggers polymerization and membrane binding. Therefore, CHMP2B is encapsulated in Buffer E containing 70 mM NaCl using Lipid Mix E, which does not contain any negatively charged lipids. In these conditions, CHMP2B does not interact with the membrane. Therefore, the proteins present outside the GUVs can be easily washed away by dilution (Figure 2).

The second population of GUVs contains PI(4,5)P2 in a lipid mix that mimics the composition of the inner leaflet of the plasma membrane (Prévost et al., 2015), Here, the buffer P has a low NaCl concentration, so that, upon GUV fusion, it will mix with buffer E resulting in optimal NaCl concentration to promote CHMP2B:PI(4,5)P2 interaction.

A further constrain in the buffers' composition is that the inner buffers (E and P) must be denser than the Outer buffer, to allow the GUVs to sediment both during centrifugation and in the observation chamber (Mathivet et al., 1996). Moreover, all buffers must have the same osmolarity. By using a combination of sucrose, glucose and NaCl, these conditions can be achieved (see Recipes section).

The proteins present outside the GUVs are removed by sequential dilution, and at the same time streptavidin-coated nanorods that interact with biotinylated lipids, are added (Figure 2). Once the GUVs are properly positioned, irradiation of the nanorods at the GUVs' interface will induce a strong local heating, in turn causing membrane instability and thus fusion (Rørvig-Lund et al., 2015). We improved the efficiency of this method by directly tethering the nanorods to the membrane via streptavidin:biotin interaction.


Figure 2. Stepwise procedure for protein encapsulation and washing. Two populations of GUVs are grown separately, then diluted in Outer buffer and centrifuged. GUVs collected from the bottom of the Eppendorf are added to Streptavidin-coated Gold nanorods (in yellow) and further diluted in Outer buffer before being introduced to the observation chamber together with the Streptavidin-coated microbeads (in grey).

Materials and Reagents

  1. Consumables
    1. Pipette tips
    2. 18 x 18 mm glass coverslips (Menzel, catalog number: BB018018A1)
    3. Hamilton syringe (Dominique Dutscher, Type 701/N)
    4. 3.5 mm culture dish (Sigma-Aldrich, catalog number: CLS430165)
    5. 1.5 ml centrifuge tube (Sigma-Aldrich, catalog number: T9661)
    6. 0.5 ml tube (Sigma-Aldrich, catalog number: T9036)
    7. 0.22 µm membrane filter (MF-MilliporeTM, catalog number: GSWP04700)
    8. 1 ml syringe (Sigma-Aldrich, catalog number: Z230723).
    9. Kimtech precision tissue task wipe (Kimtech, catalog number: 05511)

  2. Lipids
    Note: All lipids were purchased in powder from Avanti Polar Lipids, dissolved in chloroform (unless otherwise indicated) and stored at -20 °C.
    1. Egg L-α-phosphatidylcholine [EPC] (Avanti Polar Lipids, catalog number: 840051)
    2. 1,2-dioleoylsn-glycero-3-phospho-L-serine [DOPS] (Avanti Polar Lipids, catalog number: 840035)
    3. 1,2-dioleoyl-sn-glycero-3-phosphatidylethanol-amine [DOPE] (Avanti Polar Lipids, catalog number: 850725)
    4. Cholest-5-en-3β-ol [cholesterol] (Avanti Polar Lipids, catalog number: 700000)
    5. L-α-phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] (Avanti Polar Lipids, catalog number: 840046)
      Note: Dissolved in chloroform:methanol solution (70:30).
    6. L-α-phosphatidylethanolamine-N- (lissamine Rhodamine B sulfonyl) [egg PE–Rhodamine] (Avanti Polar Lipids, catalog number: 810146)
    7. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] [DSPE-PEG(2000)-biotin] (Avanti Polar Lipids, catalog number: 880129)

  3. Other reagents
    1. β-casein from bovine milk (> 99%) (Sigma-Aldrich, catalog number: C6905)
    2. Sucrose (Sigma-Aldrich, catalog number: S7903)
    3. D+ glucose (Sigma-Aldrich, catalog number: G7021)
    4. NaCl (Sigma-Aldrich, catalog number: S7653)
    5. Tris (Sigma-Aldrich, catalog number: 10708976001)
    6. Streptavidin-functionalized gold nanorods (NanopartzTM, catalog number: C12-10-850-TS-DIH-50)
    7. Polyvinyl alcohol (PVA; Merck, catalog number: 8.14894.0101)
    8. Polystyrene streptavidin coated beads (diameter 3.2 μm) (Spherotech, catalog number: SVP-30-5)
    9. Chloroform (Sigma-Aldrich, catalog number: 288306)
    10. Mineral oil (Sigma-Aldrich, catalog number: M5310)
    11. Ethanol (96%, Sigma-Aldrich, catalog number: 16368)
    12. Lipid mix P (see Recipes)
    13. Lipid mix E (see Recipes)
    14. Buffer P (see Recipes)
    15. Buffer E (see Recipes)
    16. Outer buffer (see Recipes)
    17. PVA solution (see Recipes)
    18. β-casein solution (see Recipes)

Equipment

The equipment has been accurately described in Prévost et al. (2017).

  1. Micropipettes: borosilicate capillaries with internal and external radii of 0.78 mm and 1 mm, respectively (Harvard apparatus, catalog number: 30-0036)
  2. Phase-contrast microscope: Nikon TE2000 inverted microscope, eC1 confocal system (Nikon), with two laser lines (λ = 488 nm and 543 nm); optical tweezers induced by a 5 W ytterbium fiber continuous wave laser (λ > 1070 nm; IPG GmBH Germany)
  3. Plasma cleaner (SPI Supplies, Plasma Prep III)
  4. Oven: Series B Classic.Line (Binder, Mode B 28)
  5. Vacuum pump: Pfeiffer Balzers Turbo Molecular Pumps DRAG 020 and Pfeiffer Balzers TCP 015 Turbo Molecular Pump Controller
  6. Glass syringe: Hamilton 701N (10 µl) (VWR, catalog number: 549-1135)

Procedure

  1. Preparation of GUVs by PVA-assisted swelling, washing and nanorods incubation
    1. Clean 18 x 18 mm glass slides by rinsing twice with ethanol and water. Dry them with Kimtech tissue. A separate slide is needed for each GUV population.
      Optional: Plasma-clean the glass slides for 5 min using air as plasma source. This facilitates PVA spreading and attachment. 
    2. Warm up the PVA solution to ~55 °C and spread 120 µl of it on the glass slides. Drain the excess of PVA by leaning the side of the slide on Kimtech tissue. PVA can also be spin-coated on the slide at 90 x g.
    3. Incubate the slides at 60 °C for 50 min in oven. Cool down to room temperature before use.
    4. Prepare lipid mixes P and E (Recipes 1 and 2) in chloroform to a total final concentration of 1 mg/ml. For multiple fusion events with different proteins or lipids, prepare additional mixes.
    5. Clean the Hamilton syringe with chloroform thoroughly and spread 15 μl of each lipid mix on each corresponding slide using the side of the needle until the chloroform has completely evaporated and the slide appears dry.
    6. Position each slide into a separate 3.5 mm culture dish and remove the residual organic solvent using a vacuum pump.
      Note: For sensitive lipids such as PI(4,5)P2, 20 min in vacuum are sufficient to remove the residual organic detergent without compromising the integrity of the lipids.
    7. Dilute the protein(s) of interest in Buffer E (Recipe 4).
      Note: Take into account the potential effect of the storage buffer on the final osmolarity and salt concentration of Buffer E.
    8. Deposit 360 μl of Buffers P and E (Recipes 3 and 4) on the corresponding slides. Avoid overflowing of the buffer out of the slide.
    9. Incubate the slides for 30-60 min at the desired temperature to allow swelling. GUV growth can be easily visualized with a phase-contrast microscope.
    10. Collect 200 μl of GUVs form each slide, by tilting the dish and gently rinsing the slide by gentle pipetting. Transfer each GUVs preparation to a separate 1.5 ml centrifuge tube.
      Note: GUVs are very fragile. Pipetting should be as gentle as possible.
    11. Add 1.2 ml of Outer buffer (Recipe 5) to each tube; gently mix by inverting the tube several times.
    12. Centrifuge for 10 min at 100 x g. The GUVs will sink to the bottom. Transfer 20 μl of GUV from the bottom of the tube to a new 0.5 ml tube. 
    13. Add 80 μl of Outer buffer and 0.8 μl of streptavidin-coated gold nanorods to the GUVs from the previous step. Mix gently by pipetting until the nanorods are uniformly suspended. Incubate for 20 min at room temperature.
      Note: The dilutions in Steps A11 and A13 lower the concentration of the protein present in Buffer E outside the GUVs and helps in removing lipid aggregates and small vesicles.

  2. Preparation of the experimental chamber
    1. Prepare the experimental chamber and micropipettes as previously described, also step-by-step by a movie (Prévost et al., 2017). At least three micropipettes are necessary to perform the experiment (Figure 3). While a precise pressure control is needed for R- and L-pipettes since each aspirates a GUV by connecting them to water reservoirs of controlled height (Kwok and Evans, 1981), the pressure of the T-pipette used to hold the microbead can be adjusted manually using a syringe. The tip diameter of the R- and L-pipette should be in the range 5-10 μm, while the T-pipette should have an opening < 3 μm. Position the tip of all three micropipettes at the center of the stage, within the field of view, ~100 μm above the bottom of the chamber.
    2. Passivate the chamber and pipettes by incubating with β-casein solution (Recipe 7) for 20 min. The volume of the chamber is ~200 μl. Remove the β-casein from the chamber, and rinse with Outer buffer. Fill the chamber with 170 μl of the buffer.
      Note: About 1 ml of β-casein solution is needed to fill both the chamber and the micropipettes.
    3. Carefully aspirate15 μl of each population of GUVs from the bottom of the Eppendorf tube and add them slowly to the chamber. Place the two populations on separate regions of the chamber (Figure 3).
    4. Add 2 μl of streptavidin-coated polystyrene beads (diameter ~3 μm) to the chamber to a final concentration of 0.1 x 10-3% (w/v). Place the beads in a separate region of the chamber, to avoid mixing with the GUVs (Figure 3).
    5. Keep the GUVs in the chamber for 20-30 min. During this time, the GUVs and the beads settle to the bottom of the chamber. Moreover, evaporation from buffer creates an osmotic gradient, which renders the GUVs “floppy” and allows their aspiration by the micropipettes.
    6. In order to prevent further evaporation during the experiment, seal the chamber with mineral oil.


      Figure 3. Schematic representing the arrangement of the experimental chamber including pipettes, GUVs and microbeads. L (left) and R (right) micropipettes hold both GUVs before fusion, whereas the T-pipette is used to firmly hold a micro-bead and pull the nanotube.

  3. GUVs fusion experiment (for movies depicting the fusion event, please see Supplementary data from De Franceschi et al., 2018):
    1. Lower the tip of the T-pipette to the bottom of the chamber. By moving the microscope stage, find a microbead and aspirate it strongly with the T-pipette. Lock the pressure by closing the valve on the tubing between pipette and syringe. Raise the tip of the T-pipette 100 μm from the bottom of the chamber.
    2. Set the R- and L-pipette aspiration pressure to zero. By moving the microscope stage, find a PI(4,5)P2-containing GUV and aspirate it with the R-pipette. Raise the GUV 100 μm above the bottom of the chamber.
    3. By moving the microscope stage, find a protein-containing GUV. Lower the L-pipette and aspirate the GUV. Raise this pipette 100 μm from the bottom of the chamber.
    4. Adjust the focus on the microbead held by the T-pipette. Release the pressure of the R-pipette to almost zero and bring it in contact with the bead. By moving the R-pipette with the micromanipulator away from the bead, pull a nanotube from the PI(4,5)P2-containing GUV. Adjust the height of the R-pipette so that both the microbeads and the nanotube are in focus.
    5. By using the micromanipulators, position laterally and vertically the R- and L-pipettes so that the infra-red laser is focused at the interface between both GUVs. Release the aspiration pressure of both R- and L-pipettes to nearly zero.
      Important: This is the most critical step, since fusion will occur only if the GUVs are placed correctly and if the aspiration pressure of both the R- and L-pipettes is nearly zero. Observing the GUVs by DIC rather than fluorescence, both during positioning and during fusion, greatly simplify this procedure (Figure 4).


      Figure 4. Positioning and fusion of GUVs. A. Fluorescent image of GUVs before and immediately after fusion. Lipid mixing is still not completed. The contrast in the image after fusion has been enhanced to show that fusion occurs without protein leaking outside the GUVs. Scale bar = 10 µm. B. DIC imaging of GUVs optimal position before fusion. The red dot indicates the position of the optical tweezers. Scale bar = 10 µm.

    6. Trigger fusion by turning on the infra-red laser. If the GUVs are aligned correctly, fusion is instantaneous. If fusion does not occur, switch off the laser and fine-tune the position of the GUVs by using the micromanipulators. Then repeat the process.
      Note: It is also possible to leave the laser on while adjusting the position of the pipettes. However, this occasionally induces strong adhesion between the two GUVs, which can inhibit fusion. Moreover, prolonged irradiation can cause membrane heating (due to nanorods absorption) and thus degradation of sensitive lipids such as PI(4,5)P2.
    7. After fusion, content mixing is almost instantaneous, while lipid mixing might require tens of seconds to be completed (Figure 4). This may cause fluctuations in membrane tension independently from the aspiration pressure of the pipettes. Wait 2-3 min until the tension stabilizes before performing any measurement. The focus may have to be readjusted in order to visualize the nanotube. There is no release of GUV content during or after fusion.
    8. Once membrane tension is stable, tube radius can be changed to study recruitment of proteins as a function of tube radius, as usually done with nanotube assays (Prévost et al., 2017).

Data analysis

The fusion protocol described here is used to reconstitute proteins inside GUVs. Once fusion has been accomplished, measurements and data analysis are performed similarly to a classical tube-pulling experiment (Prévost et al., 2017). In our setup, the optical trap (hence the infra-red laser) was used to induce fusion, and therefore force measurement was not possible. However, in a setup having multiple optical traps, force measurements can be performed.

Notes

  1. This protocol is independent of the method used to grow GUVs, but we recommend using the PVA-swelling method, given a good protein encapsulation efficiency and a high yield of GUVs. GUVs are very fragile and a significant amount of the GUVs produced are lost during the washing procedure, making other methods, such as electroformation on platinum wires (Prévost et al., 2017) less suitable, since less GUVs are produced. Moreover, PVA-assisted GUV swelling can work over a wide range of temperatures (4-60 °C) and thus can be adapted to the experimental conditions needed for essentially all proteins and lipids.
  2. It is generally easier to fuse GUVs of similar size, because they are easier to align.
  3. In doing a tube pulling experiment, it is important to make sure that the vesicles are unilamellar. The PVA-swelling method generally produces unilamellar vesicles. However, confirming the unilamellarity of vesicles is not necessary when using our protocol, since occasional fusion of multilamellar vesicles is very easy to recognize: only the outer membrane will undergo fusion, thus revealing the presence of inner membranes (Figure 5).


     Figure 5. Fusion event with a multilamellar GUV. The GUV in magenta is multilamellar, but this becomes evident only after fusion, since only the outer membrane can fuse with the other GUV. This creates an additional compartment, filled with proteins, between the membranes. Scale bar = 10 µm.

  4. There is a significant batch-to-batch variation in the degree of streptavidin-functionalization of gold nanoparticles. This can severely affect the efficiency of GUV fusion, and the amount of nanoparticles added needs to be adjusted for each new batch. However, the amount of nanoparticles that are generally supplied in each batch is sufficient for hundreds of experiments.

Recipes

Notes:

  1. The lipid mixes P and E and the P, E and Outer buffers described in this protocol are needed for a fusion experiment between the ESCRT-III protein CHMP2B and GUVs containing PI(4,5)P2. Different proteins, lipids or binding condition might require adjustment of the lipid mixes and buffers.
  2. For each GUV production, 360 μl of buffer is needed. About 3 ml of Outer buffer are needed for each experiment. Buffer composition can vary widely depending on the requirements and aims of the experiments. Always make sure that all buffers have a similar osmolarity, and that buffers P and E contain enough sucrose compared to the glucose content of the Outer buffer, in order to make the GUV sink.
  3. In this example, 23 μl of CHMP2B stock solution at 30 μM is added to 337 μl of Buffer E. The protein stock buffer contains 100 mM NaCl and 25 mM HEPES, thus it does not significantly affect the osmolarity.

  1. Lipid mix P 

  2. Lipid mix E

  3. Buffer for PI(4,5)P2-containing GUVs (Buffer P)

  4. Encapsulation buffer (Buffer E)

  5. Outer buffer

  6. PVA solution
    1. Dissolve 5% w/v of PVA in the following buffer: 100 mM Sucrose, 50 mM NaCl and 50 mM Tris pH 7.4
    2. Dissolve PVA by gentle stirring at ~80 °C
  7. β-casein solution
    1. Dissolve β-casein at 5 mg/ml in the following buffer: 100 mM NaCl and 50 mM Tris pH 7.4
    2. Filter the solution with a 0.22 µm membrane filter

Acknowledgments

This work was supported by the Agence Nationale de la Recherche (ANR-14-CE09-0003-01). Post-doctoral fellowships from the Institut Curie, the Fondation pour la Recherche Médicale (SPF20160936338), European Molecular Biology Organization (EMBO) non-stipendiary long term fellowship (ALTF 818-2016), MSCA Grant 751715 (ESCRT model), are acknowledged for funding the development of this method and its dissemination.
  This method, first employed in De Franceschi et al. (2018), combines and optimizes three techniques: classic tube pulling assay (Prévost et al., 2017), laser-triggered GUV fusion (Rørvig-Lund et al., 2015) and protein encapsulation by PVA-assisted gel swelling (Weinberger et al., 2013).

Competing interests

The authors declare no conflict of interest.

References

  1. Caillat, C., Maity, S., Miguet, N., Roos, W. H. and Weissenhorn, W. (2019). The role of VPS4 in ESCRT-III polymer remodeling. Biochem Soc Trans 47(1): 441-448.
  2. Dasgupta, R. and Dimova, R. (2014). Inward and outward membrane tubes pulled from giant vesicles. J Phys D Appl Phys 47(28): 282001.
  3. De Franceschi, N., Alqabandi, M., Miguet, N., Caillat, C., Mangenot, S., Weissenhorn, W. and Bassereau, P. (2018). The ESCRT protein CHMP2B acts as a diffusion barrier on reconstituted membrane necks. J Cell Sci 132(4).
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简介

蛋白质与正弯曲膜之间相互作用的体外研究可以使用经典的纳米管拉伸方法进行。 然而,表征蛋白质与负弯曲膜的相互作用仍然是一项艰巨的挑战。 在这里,我们描述了我们最近开发的基于激光触发的巨单层囊泡(GUV)融合的方法。 我们的方案允许将蛋白质顺序添加到负弯曲的膜中,同时控制缓冲液组成,脂质组成和膜张力。 此外,该方法不需要蛋白质分离步骤,大大简化了蛋白质包封相对于现有方法的过程。
【背景】包括囊泡运输在内的细胞膜重塑过程可以在拓扑上分裂成外向内出芽,例如内吞作用和内向外出芽,例如早期内体中的腔内囊泡形成,包膜病毒出芽和胞质分裂。萌芽事件引起膜形状的变化,导致两种不同的拓扑结构:凸面或凹面,通常分别定义为正曲率和负曲率(图1)。

体外方法用于研究基于膜纳米管拉伸的外向萌芽过程,例如在胞吞作用期间蛋白质与正弯曲膜的相互作用已经确立(Sorre et al。 ,2012;Prévost et al。,2017)。相反,研究蛋白质与负弯曲膜的相互作用更具挑战性,因为通常不能直接获得这样的拓扑结构 - 即,将蛋白质结合到经典GUV中拉出的膜纳米管中拉管实验。实际上,膜必须远离存在蛋白质的隔室变形(图1)。将感兴趣的蛋白质包封在GUV内部原则上可以在GUV溶胀期间实现,并且之后通过拉出纳米管来实现(Prévost et al。,2015)。然而,该方法需要将蛋白质从GUV的外部小叶上分离,这只能在结合是可逆的情况下进行,并且必须针对每种单独的蛋白质调整程序。此外,当蛋白质被包封时,它在肿胀期间也与膜相互作用,可能妨碍自身膨胀的过程。该方案的另一个限制是不可能顺序添加蛋白质(或脂质)。另一种方法包括在GUV内拉出纳米管,并通过微量移液器注射从外部添加蛋白质(Dasgupta和Dimova,2014)。但是这种方法需要非常松弛的GUV,流动和光学镊子的组合以形成管,并且当管直径必须控制到15-20nm时不适合。

在这里,我们描述了一个协议,以研究蛋白质与负弯曲膜和膜颈的相互作用(De Franceschi et al。,2018)。我们应用这种方法来研究运输所需的内体分选复合物(ESCRT)机器,它通过在带负电荷的负弯曲膜上聚合来催化内外膜出芽过程(Henne et al。, 2013; McCullough et al。,2018; Caillat et al。,2019)。表征ESCRT-III复合物特别具有挑战性,因为这些蛋白质表现出与膜的强结合,因此在包封过程后从GUV的外膜上除去它们是不可能的(De Franceschi et al。 ,2018),使其他方法(Prévost et al。,2015)不合适。我们的方法依赖于激光触发的GUV融合(Rørvig-Lund et al。,2015),并且与现有方法相比具有许多优点。首先,蛋白质包封在抑制蛋白质与膜结合的条件下进行,避免抑制GUV肿胀和蛋白质/脂质聚集。因此,可以使用缓冲液包封蛋白质,使其保持稳定并避免不希望的本体聚合。然而,在与不含蛋白质的GUV融合后,可以实现最佳结合条件,包括脂质种类和盐浓度。重要的是,在拉伸膜纳米管之后触发GUV融合,因此允许蛋白质与负弯曲膜拓扑结构的动态相互作用可视化。此外,通过进行多个融合事件,可以顺序添加额外的蛋白质。类似地,脂质组合物可以通过顺序融合事件以受控方式调节。


图1.表示从外到内和外向外萌芽事件的示意图,以及在这些过程中产生的膜曲率

方法概述: 此方法的原理是将蛋白质置于一个中性GUV(图2中的绿色)和第二个不含蛋白质的GUV(由带负电的脂质(洋红色)制成)在图2中)直到触发GUV融合。预先从带负电荷的GUV(品红色)拉出管,使得在GUV融合时,蛋白质可获得负膜曲率。因此,绿色GUV中的脂质混合物和缓冲液被设计用于维持蛋白质的稳定性并允许其包封以避免过早结合和聚集。同时,由GUV融合产生的最终脂质和缓冲液组合物必须为蛋白质 - 膜相互作用创造适当的条件。我们通过解释用于研究ESCRT-III蛋白CHMP2B和PI(4,5)P2之间结合的缓冲液和脂质混合物的设计来举例说明这一概念(De Franceschi et al。,2018)。

CHMP2B在相对高的盐浓度下稳定。在带负电荷的脂质如PI(4,5)P2存在下将NaCl浓度降低至50mM会引发聚合和膜结合。因此,使用脂质混合物E将CHMP2B包封在含有70mM NaCl的缓冲液E中,其不含任何带负电的脂质。在这些条件下,CHMP2B不与膜相互作用。因此,存在于GUV外部的蛋白质可以通过稀释容易地洗掉(图2)。

第二组GUV含有脂质混合物中的PI(4,5)P2,其模拟质膜内部小叶的组成(Prévost et al。,2015)。这里,缓冲液P具有低NaCl浓度,因此,在GUV融合时,它将与缓冲液E混合,产生最佳NaCl浓度,以促进CHMP2B:PI(4,5)P2相互作用。

缓冲液组成的进一步限制是内部缓冲液(E和P)必须比外部缓冲液更密集,以使GUV在离心和观察室中沉淀(Mathivet 等。,1996)。而且,所有缓冲液必须具有相同的渗透压。通过使用蔗糖,葡萄糖和NaCl的组合,可以实现这些条件(参见食谱部分)。

存在于GUV外部的蛋白质通过连续稀释除去,同时添加与生物素化脂质相互作用的链霉抗生物素蛋白包被的纳米棒(图2)。一旦GUV正确定位,在GUV的界面处照射纳米棒将引起强烈的局部加热,进而导致膜不稳定并因此导致融合(Rørvig-Lund 等人,,2015)。我们通过链霉抗生物素蛋白:生物素相互作用将纳米棒直接束缚到膜上,从而提高了该方法的效率。


图2.蛋白质包封和洗涤的逐步程序。分别培养两个GUV群体,然后在外部缓冲液中稀释并离心。将从Eppendorf底部收集的GUV加入到链霉抗生物素蛋白包被的金纳米棒(黄色)中,并在外部缓冲液中进一步稀释,然后与链霉抗生物素蛋白包被的微珠(灰色)一起引入观察室。

关键字:GUV, 膜融合, 负弯曲的膜, ESCRT, CHMP, 自下而上, 蛋白质包封, 纳米管牵引

材料和试剂

  1. 耗材
    1. 移液器吸头
    2. 18 x 18 mm玻璃盖玻片(Menzel,目录号:BB018018A1)
    3. Hamilton注射器(Dominique Dutscher,701 / N型)
    4. 3.5毫米培养皿(Sigma-Aldrich,目录号:CLS430165)
    5. 1.5 ml离心管(Sigma-Aldrich,目录号:T9661)
    6. 0.5毫升管(Sigma-Aldrich,目录号:T9036)
    7. 0.22μm膜过滤器(MF-Millipore TM ,目录号:GSWP04700)
    8. 1ml注射器(Sigma-Aldrich,目录号:Z230723)。
    9. Kimtech精密组织任务擦拭(Kimtech,目录号:05511)

  2. 脂质
    注意:所有脂质均购自Avanti Polar Lipids粉末,溶于氯仿(除非另有说明),并在-20°C下保存。
    1. 蛋L-α-磷脂酰胆碱[EPC](Avanti Polar Lipids,目录号:840051)
    2. 1,2-二油酰基 - 甘油-3-磷酸-L-丝氨酸[DOPS](Avanti Polar Lipids,目录号:840035)
    3. 1,2-二油酰-sn-glycero-3-phosphatidylethanol-amine [DOPE](Avanti Polar Lipids,目录号:850725)
    4. Cholest-5-en-3β-ol [胆固醇](Avanti Polar Lipids,目录号:700000)
    5. L-α-磷脂酰肌醇-4,5-二磷酸[PI(4,5)P2](Avanti Polar Lipids,目录号:840046)
      注意:溶于氯仿:甲醇溶液(70:30)。
    6. L-α-磷脂酰乙醇胺-N-(丽丝胺罗丹明B磺酰基)[蛋PE-罗丹明](Avanti Polar Lipids,目录号:810146)
    7. 1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-N- [生物素(聚乙二醇)-2000] [DSPE-PEG(2000)-biotin](Avanti Polar Lipids,目录号:880129)

  3. 其他试剂
    1. 来自牛奶的β-酪蛋白(> 99%)(Sigma-Aldrich,目录号:C6905)
    2. 蔗糖(Sigma-Aldrich,目录号:S7903)
    3. D +葡萄糖(Sigma-Aldrich,目录号:G7021)
    4. NaCl(Sigma-Aldrich,目录号:S7653)
    5. Tris(Sigma-Aldrich,目录号:10708976001)
    6. 链霉抗生物素蛋白功能化金纳米棒(Nanopartz TM ,目录号:C12-10-850-TS-DIH-50)
    7. 聚乙烯醇(PVA; Merck,目录号:8.14894.0101)
    8. 聚苯乙烯链霉抗生物素蛋白包被珠(直径3.2μm)(Spherotech,目录号:SVP-30-5)
    9. 氯仿(Sigma-Aldrich,目录号:288306)
    10. 矿物油(Sigma-Aldrich,目录号:M5310)
    11. 乙醇(96%,Sigma-Aldrich,目录号:16368)
    12. 脂质混合物P(见食谱)
    13. 脂质混合物E(见食谱)
    14. 缓冲液P(见食谱)
    15. 缓冲液E(见食谱)
    16. 外部缓冲区(见食谱)
    17. PVA解决方案(见食谱)
    18. β-酪蛋白溶液(见食谱)

设备

该设备已在Prévost et al。(2017)中准确描述。

  1. 微量移液管:内部和外部半径分别为0.78 mm和1 mm的硼硅酸盐毛细管(哈佛设备,目录号:30-0036)
  2. 相差显微镜:尼康TE2000倒置显微镜,eC1共聚焦系统(尼康),有两条激光线(λ= 488 nm和543 nm);由5W镱光纤连续波激光器(λ> 1070nm; IPG GmBH德国)引起的光学镊子
  3. 等离子清洁剂(SPI Supplies,Plasma Prep III)
  4. 烤箱:B系列Classic.Line(活页夹,模式B 28)
  5. 真空泵:Pfeiffer Balzers涡轮分子泵DRAG 020和Pfeiffer Balzers TCP 015 Turbo分子泵控制器
  6. 玻璃注射器:Hamilton 701N(10μl)(VWR,目录号:549-1135)

程序

  1. 通过PVA辅助溶胀,洗涤和纳米棒孵育制备GUV
    1. 用乙醇和水冲洗两次,清洁18 x 18 mm载玻片。用Kimtech纸巾擦干它们。每个GUV人群都需要单独的幻灯片。
      可选:使用空气作为等离子源等离子清洁玻璃载玻片5分钟。这有利于PVA的传播和附着。&nbsp;
    2. 将PVA溶液预热至约55°C并将120μl溶液涂布在载玻片上。通过将载玻片的侧面倾斜在Kimtech组织上来排出过量的PVA。 PVA也可以在载玻片上以90 x g旋涂。
    3. 将载玻片在60℃下在烘箱中孵育50分钟。使用前冷却至室温。
    4. 在氯仿中制备脂质混合物P和E(配方1和2)至总终浓度为1mg / ml。对于具有不同蛋白质或脂质的多个融合事件,准备额外的混合物。
    5. 用氯仿彻底清洗Hamilton注射器,并使用针的侧面将15μl每种脂质混合物涂在每个相应的载玻片上,直到氯仿完全蒸发并且载玻片看起来干燥。
    6. 将每个载玻片放入单独的3.5 mm培养皿中,并使用真空泵除去残留的有机溶剂。
      注意:对于敏感的脂质,如PI(4,5)P2,在真空中20分钟就足以去除残留的有机洗涤剂而不会影响脂质的完整性。
    7. 稀释缓冲液E中的目标蛋白质(配方4)。
      注意:考虑到储存缓冲液对缓冲液E的最终渗透压和盐浓度的潜在影响。
    8. 在相应的载玻片上沉积360μl缓冲液P和E(配方3和4)。避免缓冲区溢出幻灯片。
    9. 将载玻片在所需温度下孵育30-60分钟以使肿胀。使用相差显微镜可以容易地观察到GUV生长。
    10. 通过倾斜培养皿并通过轻轻移液轻轻冲洗载玻片,从每个载玻片收集200μlGUV。将每个GUV制剂转移到单独的1.5ml离心管中。
      注意:GUV非常脆弱。移液应尽可能温和。
    11. 每管加入1.2毫升外缓冲液(配方5);通过反转管轻轻混合几次。
    12. 在100 x g 下离心10分钟。 GUV将沉到底部。将20μlGUV从试管底部转移到新的0.5 ml管中。&nbsp;
    13. 将80μl外部缓冲液和0.8μl链霉抗生物素蛋白包被的金纳米棒添加到上一步的GUV中。通过移液轻轻混合直至纳米棒均匀悬浮。在室温下孵育20分钟。
      注意:步骤A11和A13中的稀释液降低了GUV外缓冲液E中存在的蛋白质浓度,有助于去除脂质聚集体和小囊泡。

  2. 制备实验室
    1. 如前所述准备实验室和微量移液管,也是电影一步一步准备(Prévost et al。,2017)。至少需要三个微量移液管才能进行实验(图3)。虽然R型和L型移液器需要精确的压力控制,因为每个人通过将它们连接到受控高度的水库来吸入GUV(Kwok和Evans,1981),用于固定微珠的T型移液管的压力可以是使用注射器手动调节。 R-和L-移液管的尖端直径应在5-10μm的范围内,而T-移液管的开口应小于5-10μm。 3微米。将所有三个微量移液管的尖端定位在平台的中心,在视野内,在腔室底部上方约100μm。
    2. 通过与β-酪蛋白溶液(配方7)一起温育20分钟来钝化腔室和移液管。腔室的体积约为200μl。从腔室中取出β-酪蛋白,并用外部缓冲液冲洗。用170μl缓冲液填充腔室。
      注意:需要约1 ml的β-酪蛋白溶液来填充腔室和微量移液器。
    3. 从Eppendorf管的底部小心地吸取15μl每种GUV群,并将它们缓慢加入室中。将两个群体放在室的不同区域(图3)。
    4. 向腔室中加入2μl链霉抗生物素蛋白包被的聚苯乙烯珠粒(直径~3μm)至终浓度为0.1×10 5 sup / 3%(w / v)。将珠子放在腔室的单独区域,以避免与GUV混合(图3)。
    5. 将GUV保持在腔室中20-30分钟。在此期间,GUV和珠子沉降到腔室的底部。此外,从缓冲液中蒸发产生渗透梯度,这使得GUV“松软”并允许它们通过微量移液管抽吸。
    6. 为了防止实验过程中的进一步蒸发,用矿物油密封腔室。


      图3.表示实验室布置的示意图,包括移液器,GUV和微珠。 L(左)和R(右)微量移液管在融合前固定两个GUV,而T型移液管用于牢固地固定微珠并拉动纳米管。

  3. GUVs融合实验(对于描述融合事件的电影,请参阅De Franceschi的补充数据 et al。,2018):
    1. 将T形移液管的尖端降低到腔室的底部。通过移动显微镜载物台,找到微珠并用T型移液管强力吸出。通过关闭移液管和注射器之间管道上的阀门来锁定压力。将T形移液管的尖端从腔室底部抬起100μm。
    2. 将R-和L-移液管吸气压力设置为零。通过移动显微镜载物台,找到含有PI(4,5)P2的GUV并用R-移液管吸出。将GUV升高至腔室底部上方100μm。
    3. 通过移动显微镜载物台,找到含有蛋白质的GUV。降低L型移液管并吸出GUV。将该移液管从腔室底部抬起100μm。
    4. 调整T型移液器固定的微珠上的焦点。将R-移液管的压力释放到几乎为零并使其与珠子接触。通过用微操纵器移动R-移液管远离珠子,从含PI(4,5)P2的GUV中拉出纳米管。调整R-移液管的高度,使微珠和纳米管都聚焦。
    5. 通过使用显微操纵器,横向和垂直放置R型和L型移液器,使红外激光聚焦在两个GUV之间的界面上。释放R型和L型吸管的吸气压力几乎为零。
      重要提示:这是最关键的一步,因为只有在正确放置GUV并且R型和L型移液器的抽吸压力几乎为零时才会发生融合。在定位和融合期间,通过DIC观察GUV而不是荧光,大大简化了这一过程(图4)。


      图4. GUV的定位和融合。 A. GUV在融合前和融合后的荧光图像。脂质混合仍未完成。融合后图像中的对比度已增强,表明融合发生时GUVs外没有蛋白质泄漏。比例尺=10μm。 B.融合前GUV最佳位置的DIC成像。红点表示光学镊子的位置。比例尺=10μm。

    6. 通过打开红外激光触发融合。如果GUV正确对齐,融合是瞬时的。如果没有发生融合,请关闭激光器并使用微操纵器微调GUV的位置。然后重复这个过程。
      注意:也可以在调整移液器位置的同时保持激光打开。然而,这偶尔会引起两个GUV之间的强粘附,这可以抑制融合。此外,长时间照射会导致膜加热(由于纳米棒的吸收),从而降解敏感脂质,如PI(4,5)P2。
    7. 融合后,内容物混合几乎是瞬间的,而脂质混合可能需要几十秒才能完成(图4)。这可能导致膜张力的波动,而与吸移管的抽吸压力无关。等待2-3分钟,直到张力稳定,然后再进行任何测量。可能必须重新调整焦点以使纳米管可视化。在融合期间或之后没有GUV内容的释放。
    8. 一旦膜张力稳定,可以改变管半径以研究蛋白质的募集作为管半径的函数,如通常用纳米管测定法所做的那样(Prévost等人,,2017)。

数据分析

这里描述的融合方案用于重建GUV内的蛋白质。一旦完成融合,就进行测量和数据分析,类似于经典的拉管实验(Prévost et al。,2017)。在我们的设置中,光阱(因此是红外激光)用于诱导融合,因此不可能进行力测量。然而,在具有多个光阱的设置中,可以执行力测量。

笔记

  1. 该方案与用于生长GUV的方法无关,但我们建议使用PVA溶胀方法,因为其具有良好的蛋白质包封效率和高产率的GUV。 GUV非常脆弱,并且在洗涤过程中产生了大量产生的GUV,使其他方法(例如铂丝上的电铸(Prévost et al。,2017)不太合适,因为GUV较少生产。此外,PVA辅助的GUV溶胀可以在很宽的温度范围内(4-60℃)起作用,因此可以适应基本上所有蛋白质和脂质所需的实验条件。
  2. 通常更容易融合相似尺寸的GUV,因为它们更容易对齐。
  3. 在进行管拉动实验时,重要的是确保囊泡是单层的。 PVA溶胀方法通常产生单层囊泡。然而,当使用我们的方案时,确认囊泡的单层性是不必要的,因为多层囊泡的偶然融合很容易识别:只有外膜将经历融合,从而揭示内膜的存在(图5)。 >

    &nbsp; 图5.具有多层GUV的融合事件。洋红色的GUV是多层的,但这只在融合后变得明显,因为只有外膜可以与其他GUV融合。这在膜之间产生了一个充满蛋白质的额外隔室。比例尺=10μm。

  4. 金纳米粒子的链霉抗生物素蛋白功能化程度存在显着的批次间差异。这会严重影响GUV融合的效率,并且需要针对每个新批次调整添加的纳米颗粒的量。然而,每批中通常提供的纳米颗粒的量足以进行数百次实验。

食谱

注意:

  1. 脂质混合物P和E以及该方案中描述的P,E和外部缓冲液是ESCRT-III蛋白CHMP2B和含有PI(4,5)P2的GUV之间的融合实验所需的。不同的蛋白质,脂质或结合条件可能需要调整脂质混合物和缓冲液。
  2. 对于每个GUV生产,需要360μl缓冲液。每个实验需要约3ml的外部缓冲液。缓冲液组成可根据实验的要求和目的而广泛变化。始终确保所有缓冲液具有相似的渗透压,并且缓冲液P和E含有足够的蔗糖,与外部缓冲液的葡萄糖含量相比,以使GUV下沉。
  3. 在此实施例中,将23μl30μM的CHMP2B储备液加入到337μl缓冲液E中。蛋白质储备缓冲液含有100 mM NaCl和25 mM HEPES,因此它不会显着影响渗透压。

  1. 脂质混合物P / nbsp;

  2. 脂质混合物E

  3. PI(4,5)含P2的GUV缓冲液(缓冲液P)

  4. 封装缓冲区(缓冲区E)

  5. 外缓冲区

  6. PVA解决方案
    1. 在下列缓冲液中溶解5%w / v的PVA:100mM蔗糖,50mM NaCl和50mM Tris pH 7.4
    2. 在约80℃温和搅拌下溶解PVA
  7. β-酪蛋白溶液
    1. 在下列缓冲液中溶解5mg / ml的β-酪蛋白:100mM NaCl和50mM Tris pH 7.4
    2. 用0.22μm膜过滤器过滤溶液

致谢

这项工作得到了国家知识产权局(ANR-14-CE09-0003-01)的支持。来自居里研究所,SPF20160936338基金会,欧洲分子生物学组织(EMBO)非附属长期奖学金(ALTF 818-2016),MSCA Grant 751715(ESCRT模型)的博士后奖学金被认可资助这种方法的发展及其传播。
&NBSP;这种方法首次应用于De Franceschi et al。(2018),结合并优化了三种技术:经典的管拉法(Prévost et al。,2017),激光 - 引发GUV融合(Rørvig-Lund 等人,2015)和PVA辅助凝胶肿胀的蛋白质包封(Weinberger et al。,2013)。

利益争夺

作者宣称没有利益冲突。

参考

  1. Caillat,C.,Maity,S.,Miguet,N.,Roos,W。H.和Weissenhorn,W。(2019)。 VPS4在ESCRT-III聚合物重塑中的作用。 Biochem Soc Trans 47(1):441-448。
  2. Dasgupta,R。和Dimova,R。(2014)。 从巨大的囊泡中取出向内和向外的膜管。 < em> J Phys D Appl Phys 47(28):282001。
  3. De Franceschi,N.,Alqabandi,M.,Miguet,N.,Caillat,C.,Mangenot,S.,Weissenhorn,W。和Bassereau,P。(2018)。 ESCRT蛋白CHMP2B可作为重组膜颈的扩散屏障。 J Cell Sci 132(4)。
  4. Henne,WM,Stenmark,H。和Emr,SD(2013)。膜的分子机制雕刻ESCRT途径。 Cold Spring Harb Perspect Biol 5(9)。
  5. Kwok,R。和Evans,E。(1981)。 大型卵磷脂双层囊泡的热弹性。 Biophys J 35 (3):637-652。
  6. Mathivet,L.,Cribier,S。和Devaux,P.F。(1996)。 在交流电场存在下制备的巨型磷脂囊泡的形状变化和物理特性。 Biophys J 70(3):1112-1121。
  7. McCullough,J.,Frost,A。和Sundquist,W。I.(2018)。 ESCRT-III / Vps4膜重塑和裂变复合物的结构,功能和动力学。 Annu Rev Cell Dev Biol 34:85-109。
  8. Prévost,C.,Tsai,F.C。,Bassereau,P。和Simunovic,M。(2017)。 从巨大的单层囊泡中拉出膜纳米管。 J Vis Exp (130)。
  9. Prévost,C.,Zhao,H.,Manzi,J.,Lemichez,E.,Lappalainen,P.,Callan-Jones,A。和Bassereau,P。(2015)。 IRSp53检测到阴性膜弯曲和沿膜管相分离。 Nat Commun 6:8529。
  10. Rørvig-Lund,A.,Bahadori,A.,Semsey,S.,Bendix,P.M。和Oddershede,L.B。(2015)。 由光学加热的金纳米粒子触发的囊泡融合。 Nano Lett 15(6):4183-4188。
  11. Sorre,B.,Callan-Jones,A.,Manzi,J.,Goud,B.,Prost,J.,Bassereau,P。和Roux,A。(2012)。 amphiphysin与膜的曲率耦合性质取决于其结合密度。 Proc Natl Acad Sci USA 109(1):173-178。
  12. Weinberger,A.,Tsai,F.C.,Koenderink,G.H.,Schmidt,T.F.,Itri,R.,Meier,W.,Schmatko,T.,Schroder,A。和Marques,C。(2013)。 凝胶辅助形成巨大的单层囊泡。 Biophys J 105(1):154-164。
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引用:de Franceschi, N., Alqabandi, M., Weissenhorn, W. and Bassereau, P. (2019). Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion. Bio-protocol 9(13): e3294. DOI: 10.21769/BioProtoc.3294.
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