Tether pulling force measurements confirm the difference in membrane

By GUV shape instability measurement, we found that amphiphysin and SNX9 are able to deform membranes at a smaller membrane-bound protein density compared to

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endophilin. In order to verify the different curvature generation capacities of the three BAR domain proteins, we applied an alternative technique to quantify mechanical effects of these proteins on membranes.

When a lipid tether is pulled from a GUV, a pulling force proportional to the square root

of membrane tension ( f 2 2, here f is the tether pulling force, κ and σ represent the membrane’s bending rigidity and surface tension respectively) [213] that is required

to maintain the highly curved cylindrical structure. However, BAR domain containing proteins with the ability to generate/stabilize high membrane curvature will have the potential to reduce the pulling force [155, 214]. We therefore measured the tether pulling force in the presence of each protein with the assistance of an optical trap.

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Figure 7.6 The three proteins mechanically stabilize high curvature membrane tethers to different extents. This figure was obtained by Zheng Shi. (A) Representative forces required to maintain a tether pulled from bare GUVs (black, lipid composition: DOPS/DOPE/DOPC=45/30/25), GUVs in 400 nM endophilin (blue), or GUVs in 400 nM amphiphysin (red) under various membrane tensions. Solid lines are linear fits of the pulling force to tension0.5 where the slope of the linear fit can be directly related to the effective bending rigidity of the membrane. Averaged slope values from measurements on multiple GUVs correspond to effective bending rigidities of 23.6 ± 4.0 kBT for bare

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400 nM amphiphysin. (B) Representative forces required to maintain a tether pulled from bare GUVs (black, lipid composition: PI(4,5)P2/DOPS/DOPE/DOPC=5/30/30/35), GUVs

in 200 nM amphiphysin (red), or GUVs in 400 nM SNX9 (green) under various membrane tensions. Different protein concentrations were chosen here in order to reduce the difference in membrane densities of the two proteins (see Figure 7.2D, under these conditions, the resulting densities were 1050 ± 650 μm-2 for amphiphysin and 500 ± 60 μm-2

for SNX9). Solid lines are linear fits of the pulling force to tension0.5. Effective bending rigidities are 21.9 ± 6.1 kBT for bare GUVs, 18.0 ± 4.4 kBT for GUVs in 200 nM

amphiphysin, and 18.7 ± 4.3 kBT for GUVs in 400 nM SNX9. (C) Summary of the slope

(black) and intercept (blue) extracted from the linear fits of tether pulling force to tension0.5 as shown in A). (D) Summary of the slope (black) and intercept (blue) extracted from the linear fits of tether pulling force to tension0.5 as shown in B). Student t-test: N.S.: p>0.1, *p<0.05, **p<0.005, ***p<0.0005 (the tests without an associated bracket refer to comparisons with corresponding ‘Bare GUV’ data). Light error bars are SD, dark error bars are SEM. Proteins used in this measurement were endophilin N-BAR, amphiphysin FL, and SNX9 FL.

As shown in Figure 7.6A, the presence of amphiphysin significantly lowers the pulling force required to maintain the tether, consistent with the protein’s relatively strong

curvature generation capacity [155, 214]. At the same bulk concentration, endophilin affects the tether pulling force to a much weaker extent; despite the larger number of endophilin molecules that are bound to the GUV surface (Figure 7.6A). This can be

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further quantified by determining the slope and intercept of a linear fit of the f ~ 

relation. As shown in Figure 7.6C, both the slope and intercept significantly decrease in the presence of amphiphysin compared to protein free GUVs, while no significant difference can be found for endophilin covered membranes. Therefore, amphiphysin possesses a significantly larger membrane curvature stabilizing effect than endophilin.

We then carried out the same comparison between SNX9 and amphiphysin. As shown in Figure 7.6B, at comparable protein densities, SNX9 reduces the tether pulling force to a larger extent than amphiphysin. We examined this further by performing a linear fit of

the f ~  relation of the two proteins. A significantly larger reduction in the intercept value is found for SNX9 compared to amphiphysin (Figure 7.6D).

Overall, the tether pulling force measurements imply that under similar surface densities, the proteins’ abilities to stabilize high curvature membranes follow a decreasing trend

from SNX9 to amphiphysin and to endophilin. These findings are consistent with our predictions based on the EM tubulation assay, where SNX9 and amphiphysin exhibit a lower critical concentration for tubulating liposomes than endophilin.

In a low protein density regime, one can treat the proteins as a two dimensional gas and

the tether pulling force f can then be predicted by 𝑓 = 2𝜋√2𝜅_eff𝜎 − 2𝜋𝜅𝐶₀𝜙₀, where

𝜅_eff = 𝜅(1 −𝜅𝐶0_𝑎2) , C0 represents the spontaneous curvature of the membrane induced by

protein binding, a is the effective inverse compressibility of the protein on the membrane, and ϕ0 is the protein’s surface cover fraction on the GUV [155, 214]. In other words, with

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similar protein densities on the GUV, the amount of force reduction at zero tension is predicted to be directly determined by the values of C0, with 𝑓0 = −2𝜋𝜅𝐶0𝜙0. Following this, we can calculate the spontaneous curvature per molecule of each protein from the intercepts values in Figure 7.6C and 7.6D (Table 7.3).

Protein C0 (nm

-1₎

Endophilin (N-BAR) 0.028 ± 0.017

Amphiphysin (FL) 0.34 ± 0.05

SNX9 (FL) 1.03 ± 0.14

Table 7.3 Effective spontaneous curvature predicted by the tether pulling force measurements.

These values quantitatively agree with previously reported values for N-BAR domain proteins [34, 155], and with a trend agreeing with the results from the GUV shape instability measurements (Table 7.2).

7.8 Membrane curvature sensing ability is density dependent and decreases from