Figure 1.
Experimental approaches for understanding the interaction between SOD1 aggregates and cellular membranes.
(A) A cartoons showing the protein's aggregation and its interaction with a cellular membrane. (B) The SPR instrumental setup equipped with a flow cell used in an in vitro observation with an artificial SLB. (C) The inside-out configuration of the patch clamp used in an observation with a real cell membrane patch.
Figure 2.
Schematic illustration of SOD1 aggregation and its characteristics.
(A) A scheme for the aggregation of SOD1. Inset is a shape of as-prepared SOD1 aggregate. Bars, 200 nm. (B) CD spectra and (C) ANS fluorescence intensities for proteins (Red, SOD1 aggregates; orange, apo WT SOD1; blue, holo WT SOD1; grey, BSA).; All protein samples were prepared in PBS at a concentration of 0.1 mg/mL. SOD1 aggregates were taken after incubation with TFE for 5 days, and then characterized.
Figure 3.
SPR reflectance changes after the interactions between the proteins and the SLBs.
(A) SPR reflectance decreases after the interaction between SOD1 aggregates and SLB. (B) SPR reflectance increases after the interaction between apo WT SOD1 and SLB. (C) SPR reflectance increases after the interaction between BSA and SLB. All proteins were prepared in PBS at a concentration of 0.1 mg/ml and were exposed to the lipid membrane for 1 h. The time-resolved reflectance changes derived by the interaction between SLBs and injected proteins were measured using the fixed angle method.
Figure 4.
Time-resolved ThT fluorescence measurement for the incubated proteins.
(A) With DPPC lipid vesicles. (B) Without DPPC lipid vesicles. To investigate the effect of the interaction of lipid molecules with proteins on aggregation, proteins were incubated with DPPC vesicles in PBS at 37.5°C. As controls, proteins without DPPC vesicles were also incubated under the same conditions. All samples were incubated without agitation. At each time point, an aliquot of each sample was taken and the presence of aggregates was determined using ThT fluorescence assay.
Figure 5.
AFM images of resulting SLB surfaces.
(A) Original SLB layer. (B), (C), and (D) SLB layers after the interaction with SOD1 aggregates, apo SOD1, and BSA, respectively. Bars, 1 µm. All images were obtained after the SPR measurement.
Figure 6.
The formation of defects within the SLB by the interaction between the SOD1 aggregate and the SLB.
(A) (π) and (θ) AFM image and height profile of the resulting SLB surface after the exposure to the SOD1 aggregate. (ρ) and (σ) AFM image colored for remaining lipid layers (shown in pink) and height distribution of entire surface. Remaining lipid layers occupy approximately 20% of the entire surface. (B) SPR contour plots at each event. Each plot indicates before formation of SLB (black), after formation of SLB (blue), and after interaction between SLB and SOD1 aggregates (red), respectively. (C) ThT fluorescence image of the resulting SLB surface. (An inset shows clear blue ThT fluorescence for a SOD1 aggregate solution.). Bars, 100 µm. (D) Description of the formation of defects within a SLB by the SOD1 aggregate.
Figure 7.
The unregulated membrane permeability in HEK 293 cell membrane patches by the SOD1 aggregates.
The ionic permeability of HEK 293 cell membranes in response to the perfusion of proteins was measured at 10 kHz in inside-out configuration (Fig. 1C) of a patch clamp. The bath (approximately 0.15 ml) was superfused at 1 ml/min and voltage clamp experiments were performed at room temperature (22–25°C). (A–E) Electrophysiological recordings for (A) BSA, as a control, (B) Apo WT SOD1, (C) WT SOD1 aggregate, (D) Apo A4V SOD1, and (E) A4V SOD1 aggregate. Bar graphs represent the ThT fluorescence intensity of each sample solution obtained before perfusing.