Femtoliter Injection of ESCRT-III Proteins into Adhered Giant Unilamellar Vesicles

The endosomal sorting complex required for transport (ESCRT) machinery mediates membrane fission reactions that exhibit a different topology from that observed in clathrin-coated vesicles. In all of the ESCRT-mediated events, the nascent vesicle buds away from the cytosol. However, ESCRT proteins are able to act upon membranes with different geometries. For instance, the formation of multivesicular bodies (MVBs) and the biogenesis of extracellular vesicles both require the participation of the ESCRT-III sub-complex, and they differ in their initial membrane geometry before budding starts: the protein complex acts either from outside the membrane organelle (causing inward budding) or from within (causing outward budding). Several studies have reconstituted the action of the ESCRT-III subunits in supported bilayers and cell-sized vesicles mimicking the geometry occurring during MVBs formation (in-bud), but extracellular vesicle budding (out-bud) mechanisms remain less explored, because of the outstanding difficulties encountered in encapsulation of functional ESCRT-III in vesicles. Here, we provide a different approach that allows the recreation of the out-bud formation, by combining giant unilamellar vesicles as a membrane model and a microinjection system. The vesicles are immobilized prior to injection via weak adhesion to the chamber coverslip, which also ensures preserving the membrane excess area required for budding. After protein injection, vesicles exhibit outward budding. The approach presented in this work can be used in the future to disentangle the mechanisms underlying ESCRT-III-mediated fission, recreating the geometry of extracellular bud production, which remains a challenge. Moreover, the microinjection methodology can be also adapted to interrogate the action of other cytosolic components on the encapsulating membranous organelle.

This protocol was validated in: PLOS Pathog (2021), DOI: 10.1371/journal.ppat.1009455 The endosomal sorting complex required for transport (ESCRT) machinery mediates membrane fission reactions that exhibit a different topology from that observed in clathrin-coated vesicles. In all of the ESCRT-mediated events, the nascent vesicle buds away from the cytosol. However, ESCRT proteins are able to act upon membranes with different geometries. For instance, the formation of multivesicular bodies (MVBs) and the biogenesis of extracellular vesicles both require the participation of the ESCRT-III sub-complex, and they differ in their initial membrane geometry before budding starts: the protein complex acts either from outside the membrane organelle (causing inward budding) or from within (causing outward budding). Several studies have reconstituted the action of the ESCRT-III subunits in supported bilayers and cell-sized vesicles mimicking the geometry occurring during MVBs formation (in-bud), but extracellular vesicle budding (out-bud) mechanisms remain less explored, because of the outstanding difficulties encountered in encapsulation of functional ESCRT-III in vesicles. Here, we provide a different approach that allows the recreation of the out-bud formation, by combining giant unilamellar vesicles as a membrane model and a microinjection system. The vesicles are immobilized prior to injection via weak adhesion to the chamber coverslip, which also ensures preserving the membrane excess area required for budding. After protein injection, vesicles exhibit outward budding. The approach presented in this work can be used in the future to disentangle the mechanisms underlying ESCRT-III-mediated fission, recreating the geometry of extracellular bud production, which remains a challenge. Moreover, the microinjection methodology can be also adapted to interrogate the action of other cytosolic components on the encapsulating membranous organelle.

Published: Feb 20, 2022
activates PfVps32 and PfVps60, both ESCRT-III members, triggering EV biogenesis . Involving an intracellular parasite, the study of this process is problematic. Moreover, the knockdown or deletion of ESCRT genes in other organisms results in the formation of aberrant structures that lack ILVs (Doyotte et al., 2005;Nickerson et al., 2006). To address these difficulties, membrane models have been widely used to analyze in vitro ESCRT-III-mediated events. In this direction, giant unilamellar vesicles (GUVs) (Dimova and Marques, 2019;Dimova, 2019) combined with recombinant ESCRT proteins have become an established platform to examine the formation of MVBs (Im et al., 2009;Avalos-Padilla et al., 2018 and2021a;Booth et al., 2019;Alqabandi et al., 2021). To mimic the geometry occurring in this process, ESCRT components are introduced in the vesicle surroundings. The proteins induce membrane invaginations towards the vesicle interior, which can lead to the formation of ILVs, connected to the mother membrane through a thin neck, and the final cleavage of the neck results in the formation of MVB-like GUVs.
The out-budding processes (as in the formation of microvesicles shed by the plasma membrane) exhibit a reverse budding topology, compared to that of MVB formation. Thus, to explore such processes, the ESCRT units have to act from within the vesicle model. In other words, the proteins have to be introduced into the GUVs' lumen. One approach to accomplish this consists of encapsulating ESCRT proteins inside GUVs, by forming the vesicles in the presence of the proteins; using this strategy, nanotubes with the correct topology for scission were pulled and subsequently cleaved (Schöneberg et al., 2018). However, under these conditions, one cannot observe the vesicle response upon immediate interaction with the proteins, as it relies on ATP photo-uncaging. To evade these drawbacks, we have designed an approach in which pre-formed GUVs encapsulating the buffer necessary for protein activity are injected with the ESCRT-III proteins. With this technique, we are able to observe in real time the dynamics of out-bud formation (mimicking the process driven during EV biogenesis), and to evaluate the effects specific to a particular protein.
Injection approaches in GUVs have been applied previously (Wick et al., 1996;Hurtig and Orwar, 2008;Lefrançois et al., 2018). In isolated GUVs, it is important to ensure control over the vesicle volume and area. In particular, the injection of isotonic solutions can pull out the excess membrane area needed for deformation, which would then prohibit outward budding. Thus, we adapted this protocol, by employing osmolarities of the injected solutions which lead to vesicle deflation, to create excess vesicle area for deformation and budding. Furthermore, isolated vesicles need to be immobilized to facilitate puncturing the membrane without displacing and losing them. In previous work, the immobilization was ensured by working with GUVs which were directly formed on the substrate for GUV swelling. However, such vesicles are typically connected to other GUVs and structures, thus not ensuring area/volume conservation. Here, we employed biotin-avidin-based adhesion, using biotinylated lipids in the vesicle and an avidin-coated substrate to which the GUVs were fixed as proposed earlier . Successful injection of ESCRTs and further registration of the functionality of the proteins, namely formation of out-buds, requires fine adjustment of the adhesion level. On one hand, strong adhesion stabilizes the vesicles during the puncturing procedure, while on the other hand, it increases the membrane tension while consuming the area available for deformation. The latter effect limits the ability of the membrane to bend and thus hinders budding. We thus optimized the avidin surface concentration to ensure mild adhesion. Another important aspect to consider is the buffer in which proteins remain active: in the case of ESCRT-III proteins, the buffer used was 150 mM NaCl and 25 mM Tris-HCl, pH 7.4 (~325 mOsmol/kg). This high salt concentration hampered GUVs growing through the standard electroformation protocol (Angelova and Dimitrov, 1986). Despite some recent developments of this protocol aiming at application to solutions of high ionic strength (Montes et al., 2007;Pott et al., 2008;Li et al., 2016;Lefrançois et al., 2018), it is clear that the incorporation of negatively charged lipids imposes strong limitations, resulting in poor vesicle quality and small size (as confirmed by our own tests, data not shown). To overcome these drawbacks, we used the gel-assisted method (Weinberger et al., 2013), in which we were able to grow vesicles encapsulating the protein buffer. It must be noted that this method may lead to the incorporation of polymers into the formed vesicles, thus altering their mechanical properties (Dao et al., 2017). Indeed, occasionally we observed vesicles with denser content (presumably resulting from encapsulated hydrogel). For the work here, we always selected "clean" GUVs with no visible alterations in their membranes and, as detailed later, substantiate the results with control experiments where vesicles were injected with protein-free buffer. As we are working with P. falciparum ESCRT-III subunits, the GUV lipid composition was selected to mimic the inner leaflet of the red blood cell plasma membrane (Virtanen et al., 1998). However, this can be modified depending on the system. We also demonstrate the injection and outward budding process for GUVs injected with another ESCRT-III system, Published: Feb 20, 2022 namely the protozoan parasite responsible for amoebiasis, Entamoeba histolytica, whose characterization in GUVs has been previously reported , using a suitable membrane composition. Finally, as mentioned above, to deflate the GUVs, and thus to ensure that the vesicles exhibit excess membrane area needed for the formation of the out-buds, the injected proteins were kept in a 0.8× buffer (prepared by a direct dilution in Milli-Q water of the 1× protein buffer, see Recipe 4). As a control, upon injection of GUVs with the same volume and osmolarity of protein-free solution as in the experimental set-up, no out-buds appeared, demonstrating the validity of our approach.

A. Lipids
Note: The purification of the P. falciparum proteins (PfBro1 and PfVps32) and E. histolytica proteins (EhVps20t and EhVps32) followed a conventional protocol of affinity purification using the GST-tag present in the recombinant proteins and GSH-Sepharose 4B for its capture. The GST-tag was later removed, and recombinant proteins were purified by size exclusion chromatography as detailed in  and Avalos-Padilla et al, (2018), respectively.

Procedure
The complete protocol is summarized in a flow chart shown in Figure 1. A. Formation of GUVs by PVA gel-assisted swelling 1 Recipes 2 and 3) in chloroform at 1 mg·mL -1 final concentration. 5. Clean thoroughly a Hamilton syringe with chloroform and spread 10 to 15 µL of the lipid mixture on the PVA film dried on the glass (taken from the oven without cooling it down) using the needle of the syringe and until the slide appears dry. 6. Place the glass slide for 1 h under vacuum to eliminate the excess chloroform. 7. Glue the Teflon spacer (via silicone grease) to the glass with the dried lipid film (see Figure 2A). 8. Fill the chamber with 1,800 µL of 1× protein buffer (see Recipe 4) and place a glass coverslip on top of the Teflon spacer (see Figure 2B) to avoid unwanted evaporation.

Figure 2. GUV chamber for gel-assisted swelling.
The Teflon spacer is between two cover glasses (seen in panel B). The bottom glass is coated with PVA where lipids are deposited. The white oval roughly indicates the area with the deposited lipid mixture.
9. Incubate for 10 min at room temperature, to allow swelling and GUV formation. 10. After this time, tap gently a few times on the bottom of the growing chamber, remove the upper coverslip by sliding it to the side and collect the GUVs using a 1,000 µL pipette tip without touching the PVA film, to avoid collecting PVA debris. Collected GUVs must be placed in a glass container, protected from light to avoid oxidation, and stored at room temperature (~21°C). The solutions should be used fresh, even though no differences in the behavior were observed for GUV solutions used on the following day after preparation.

B. Fabrication and loading of the micropipette
1. Take a borosilicate capillary and carefully apply N2 flow through it, to make sure that the capillary is not clogged. 2. Place the capillary at the holder of the micropipette puller ( Figure 3). 3. Pull the capillary using the one-line program, to achieve a bee-needle orifice of ~250 nm (HEAT Ramp; PULL 100, VEL 10, TIME 250, PRESSURE 500´) in the micropipette puller.    Figure 5).

Published: Feb 20, 2022
3. Attach the holder with the pipette to the mechanical arm of the micromanipulator. 4. Set the angle of injection as large as the microscope setup allows (see Figure 7).

Figure 7. Micropipette setup of the injection procedure.
A: Top-view of the setup; for clarity the observation chamber was removed. B: A close-up side-view of the injection angle. Note that the micropipette holder is as close to the condenser of the microscope as possible to ensure high angle of injection.

With the micromanipulator, introduce the micropipette into the solution of the observation chamber and
focus on the tip of the pipette. 6. Set the microscope to the desired observation settings. For the measurement presented here, they were the following: • The DPPE-Rhodamine dye (integrated in the membrane of the GUVs) was excited with a 561 nm diode-pumped solid-state laser, and the signal was collected in the 570-650 nm range. • The PEG-FITC dye was excited with a 488 nm argon laser, and the signal was collected in the 495-530 nm range. • To avoid crosstalk between the different fluorescence signals, sequential scanning was performed. 7. Place the micropipette in a site where no GUVs are observed and purge it (by pressing the "Clean" bottom of the microinjector) to confirm that the micropipette is not clogged; a signal from the fluorescent dye leaving the pipette tip should be detected. 8. Lower the micropipette close to, but still above the focal plane of the GUVs. 9. Approach a selected GUV with the tip of the micropipette from above; the selected vesicle should be clean and without defects (to ensure this, examine the selected GUV with a XYZ scan). 10. Lift the micropipette a few micrometers above the vesicle (the micropipette tip goes out of focus). 11. Puncture the GUV, by moving the micropipette towards the vesicle in both Z and X directions. 12. Perform a XYZ scan to make sure that the micropipette penetrates into the GUV. 13. Start recording a time sequence. 14. Inject the vesicle (see Figure 8 and Figure 9) using the following parameters of the microinjector: pressure Note: The ideal angle of injection would be 90°, to avoid lateral displacement of the vesicles during puncture attempts. However, this cannot be achieved since the condenser of the microscope occupies the space above the sample.
Published: Feb 20, 2022 of injection:150 hPa; time of injection: 5 s; compensation pressure: 1 hPa. These parameters ensure that injected volumes are in the sub-picoliter range (a few hundreds of femtoliters). 15. Pull the micropipette out from the interior of the GUV (lift the micropipette in Z direction). 16. Perform a XYZ scan to detect in which focal plane the outward buds formed (Figure 8, Figure 9). The membrane is presented in red and PEG-FITC in green. The tip of the injection pipette can be noticed on the membrane in the first two frames in each sequence. The upper row represents the control experiment (ESCRT-free buffer is injected, so outward buds were not observed). In contrast, when ESCRT-III recombinant proteins were injected, outward buds formed; see also Video 1. The arrows on the last frame point to the outward buds. Adapted from Avalos-Padilla et al. (2021b).

Video 1. Injection of a GUV with purified P. falciparum ESCRT-III proteins.
Real-time recording. The specific information describing the system is indicated in the caption of Figure 8. The membrane is shown in magenta and PEG-FITC in cyan. The tip of the injection pipette can be seen on the first two frames in each sequence. The arrows on the last frame point to the outward buds; see also Video 2. The inset in the last snapshot shows a zoomed-in view of outward buds from the same vesicle but at another XY plane.

Video 2. Injection of a GUV with purified E. histolytica ESCRT-III proteins.
Real-time recording. The specific information describing the system is indicated in the caption of Figure 9.

PVA solution (5% w/v)
a. Weigh 0.5 g PVA and place it in a glass vial. b. Add 10 mL of 1× protein buffer (to maintain osmolarity) and place a clean magnetic stirrer in the vial. c. Keep the solution in a 90°C water bath under constant stirring (300-400 rpm) until PVA dissolves completely. The solution becomes clear.