GUV shape stability assay

To investigate the membrane deformation ability of the BAR domain-containing proteins and of the crowding effect, we employed a GUV-based membrane shape stability assay based on micropipette aspiration (see Fig. 2.5A) to study the geometry changes of a single GUV when incubated with a BAR-domain-containing protein solution. The techniques involved in this assay are described below.

2.6.1 GUV transfer equipment and operations

As shown in Fig. 2.5A, separate GUV dispersion (membrane labeled with red dye) and protein solution (labeled with green dye) were prepared as described in Refs [153, 154]. Both solutions were 375 μl in volume and were diluted from stock GUV and protein

solutions to designated concentrations by using a buffer containing sucrose (400mM) : glucose (400mM) : protein buffer (20 mM HEPES, 150 mM NaCl) at a 1 : 1 : 1 ratio. This dilution buffer had an osmolarity that was approximately 20% higher than the GUV stock solution, which ensured that GUVs were sufficiently flaccid to allow for pipette aspiration. The solution conditions ensured that vesicle transfer occurred between two solutions of identical composition, except for the presence of protein in the receiving solution. Therefore, any observed changes in GUV geometry can be ascribed solely to protein binding, as opposed to any other changes in solution conditions. The preparation

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of GUV aspiration micropipettes and transfer capillary tubes was previously described [25, 147]. The micropipettes were casein-coated before use to avoid membrane adhesion to the inner micropipette walls. All experiments were carried out at room temperature.

The procedure of transferring a single GUV from the GUV dispersion into the protein solution includes the following steps [154]. First, the zero pressure of the system is carefully adjusted before aspirating GUVs. Next, the aspiration pressure is reduced to a negative value to aspirate a single GUV, and then the membrane tension of the GUV is adjusted to a desired value by adjusting the pipette aspiration pressure. Afterwards, the transfer capillary is manually moved forward to cap the aspirated GUV. The capped GUV is then removed from the GUV solution (red in Fig. 2.5A) and inserted into the protein solution (green in Fig. 2.5A), upon which the transfer capillary is manually moved backward to expose the GUV to protein solution. Finally, the protein binding and GUV shape transition process is monitored via confocal microscopy imaging as soon as the transfer capillary is removed and the GUV is exposed to protein solution (which defines t = 0). Confocal fluorescence imaging (Objective: 60x W 1.1 NA, Olympus, Center Valley, PA) was used to continuously capture protein density increase on the membrane and to follow GUV geometry changes induced by protein binding (Fig. 2.5B). Imaging was continued until the protein density on the GUV reached thermodynamic equilibrium (as defined by the absence of additional changes). The fluorescence intensities thus obtained were converted into an “equilibrium protein density”.

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Figure 2.5 GUV shape stability assay. A representative example of the amphiphysin induced GUV geometry changes in GUV shape stability assay. (A) The geometry of

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glass-pipette aspirated GUV and the process of transferring a GUV from a GUV dispersion to protein solution (the parameters used for calculation of membrane tension

are as follows:Rv: GUV radius; RP: micropipette radius; LP: projection length; ∆P:

pressure difference). A GUV was aspirated from a GUV dispersion (red) and protected with an outer capillary, then was transferred to a protein solution (green). After transfer, the capillary was removed and then GUV was exposed to proteins, after protein binding, the GUV was tubulated. (B) Time-lapsed confocal image of a transferred GUV in an Amphiphysin N-BAR domain solution. Arrows indicate tubules formed towards the outside of the GUV. Scale bar = 10 μm. (C) Plot of protein density on GUV and the apparent area of the GUV. Red dashed line indicates that the apparent area was stable (red dashed line) before the transition point (red arrow) when the area started to decrease. (D) Volume trace of the GUV example shown in (C). Volume is constant after transfer. Buffer: 7 mM HEPES, 50 mM NaCl, pH = 7.4. GUV composition: 45% DOPS + 30% DOPE + 24.5% DOPC + 0.5% Texas Red-DHPE.

2.6.2 Data processing

The geometry of the aspirated GUV and the parameters used for the calculation of membrane tension and geometry changes (GUV radius, Rv, micropipette radius, Rp,

length of pipette-aspirated vesicle fraction, i.e. the projection length, Lp, and pressure, ∆P)

are shown in Fig. 2.5A. Image J was used to measure micropipette radius and projection length, while a Matlab code was used to determine GUV radius and average fluorescence intensity on the GUV contour [154].

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Membrane tension, 𝜎, of a spherical lipid vesicle that is aspirated with a micropipette, where the projection length is longer than pipette radius and the shape of the cap is hemispherical, is determined by the following equation:

. (2.10)

The vesicle membrane area was calculated as follows (Fig. 2.5C):

. (2.11)

This area was used as an indicator of the GUV geometry change, and the GUV shape instability transition point was determined by the following procedure. To determine the transition density, we first chose several (minimally three) measurement points where the membrane area was observed to be constant (Fig. 2.5C, dashed red line), and determined the standard deviation (STD) and average value for this set of pre-transition points. In order to rigorously define a threshold for the shape transition instability, we subtracted 2*STD from that average value. We then determined the transition area (and time) from linear interpolation using the two area data points immediately above and below, respectively, of the threshold value. Likewise, the transition density was found from the transition time defined above and linear interpolation of the protein area density measurements (Fig. 2.5C, red arrow).

We also calculated the volume of the aspirated GUV as:

1 1 2( ) p v P R R    

 

 

48 3 2 4 ( ) ( ) ( ) * 3 v P P Volume t  R t R t L . (2.12) It is important to note that the GUV volume remained constant over the course of each experiment (Fig. 2.5D). This condition is essential for the interpretation of our experimental results because it ensures that protein binding does not lead to membrane pore formation, since volume changes induced by bulk flow through pores would result in concomitant projection length changes, which would interfere with our method to observe the onset of tubulation transitions.

For I-BAR proteins, unlike N-BAR proteins, tubules formed toward the interior of the GUV. To monitor inward tubulation, we further acquired time-lapsed mean fluorescence intensities in the GUV interior by defining a disk-shaped region of interest within the GUV contour and measured the mean fluorescence intensity inside of the circle defining this region of interest (Fig. 6.4).