Membrane insertion of—and membrane potential sensing by—semiconductor voltage nanosensors: Feasibility demonstration

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Science Advances  12 Jan 2018:
Vol. 4, no. 1, e1601453
DOI: 10.1126/sciadv.1601453
  • Fig. 1 Surface functionalization.

    (A) Cartoon describing design principles for rendering NR membrane protein–like properties. This functionalization will favor their stable, spontaneous insertion into the membrane with the correct orientation. (B) Peptide design for implementing (A). (C) Top view of an NR coated with peptides. Brown and orange colors depict Cys-rich and lipophilic faces of the α-helical peptide, respectively. (D) Sequence of the designed peptide. C14-CO- stands for myristoyl acid residue attached to the N-terminal amino group. (E) Wheel diagram corresponding to the α-helical part of the peptide. Color coding is the same as in (C). aa, amino acid.

  • Fig. 2 NR interaction with membrane.

    (A) CryoEM micrographs of pcNRs inserted into SUVs. Scale bars, 30 nm. (B) Schematics of possible pcNRs association with lipid bilayer: (a) properly inserted, (b) partially inserted, (c) attached in an angle, and (d) horizontally embedded. (C) Histogram of insertion geometries (a) to (d). (D) Schematic of the NR of hydrophobic length L with two hydrophilic ends of length b and radius a. The total length of the rod is then L + 2b. It is shown in a piece of membrane of thickness t. The green curves show the ends of the hydrophobic rod. The red curve denotes the center of the nanostructure, whereas the purple curve shows the intersection of the rod with the mid-plane of the membrane. (E) Model calculations (see section S7) of canting angles (θ) probability distribution for a membrane-inserted NR. Calculations for no hydrophobic mismatch (L = t = 4 nm, blue) and for significant hydrophobic mismatch (L = 6 nm, t = 4 nm; red) are shown. In both cases, the rods are terminated at both ends by hydrophilic cylinders with a length of 2 nm (details of the model are discussed in section S7).

  • Fig. 3 Delivery of pcNRs to HEK293 cells.

    (A) Fluorescence of NR-loaded GUVs. (B and C) Bright-field (B) and fluorescence (C) images of pcNR-loaded GUV fused with the cell membrane. (D) Fluorescence image of HEK293 cells stained with ANEPPS (control). (E and F) pcNRs targeted to membranes at high (E) and low (F) concentrations. Scale bars, 10 μm.

  • Fig. 4 Membrane voltage sensing of spiking HEK293 cells with pcNRs.

    (A and B) Fluorescence images of cells stained with ANEPPS (A) and pcNRs (B). (C) Spatially high-pass–filtered image of (B) used to highlight signals from individual pcNRs and remove background signals. (D and F) Temporal bandpass-filtered ΔF/F time trace of ANEPPS (D, top) and pcNRs (F, top). Each trace (D and F, bottom) shares the same color as the marked open circles in (A) or (C), respectively (see section S8). (E and G) Overlaid ΔF/F’s of seven frames around the gray dashed lines in (D) and (F). (D) and (F) have 23 and 19 thin lines in each subplot, respectively. Mean traces are shown with thicker line width. The leftmost subplots with black lines are ΔF/F of ensemble average [generated from top in (D) and (F)]. Scale bars, 10 μm.

  • Fig. 5 Voltage response of pcNRs.

    (A) Intensity trace of a single pcNR with time intervals of large ΔF/F modulation response (bursts) marked with a shaded area and asterisk (*). (B) Zoom-in of intensity trace during a burst in (A); each marker represents a two-frame average intensity during the voltage-on (green squares) and voltage-off (red dots) semi-period. (C) Histogram of the modulation responses for each aggregating burst from many pcNRs in a video. The first group (red) represents the set of patched pcNRs that exhibit the highest signal. The other three distributions represent control groups for the set of unpatched pcNRs and/or for out-of-phase modulation response. a.u., arbitrary units.

Supplementary Materials

  • Supplementary material for this article is available at

    section S1. Design of the peptide sequence for coating NRs

    section S2. Circular dichroism of the designed peptide

    section S3. Fluorescence anisotropy of pcNR-loaded vesicles

    section S4. Cell membrane staining with pcNRs

    section S5. CryoEM control: Ligand-coated NRs do not insert into vesicles’ membranes

    section S6. Endocytosis of pcNRs after 1 hour of loading

    section S7. Simulation of the energetics of the NR in the membrane

    section S8. Optical recording of ANEPPS-labeled and pcNR-labeled spiking HEK cells

    section S9. Simultaneous optical and electrical recordings in patch-clamp experiment

    fig. S1. Circular dichroism spectrum of designed peptides dissolved in octanol.

    fig. S2. Orientation-dependent AA of pcNRs in membranes of GV.

    fig. S3. Confocal cross-sections of an HEK293 cell fused with pcNR-loaded vesicles.

    fig. S4. CryoEM images of vesicles after incubation with pcNRs.

    fig. S5. Images of pcNR-loaded HEK293 cells taken 1 hour later.

    fig. S6. Canting angle distribution of NR.

    fig. S7. Image processing of voltage recording with ANEPPS.

    fig. S8. Image processing of voltage recording with pcNR.

    fig. S9. Mean of {ΔFi/F} for the two sets of patched (left) and unpatched (right) particles.

    fig. S10. Image processing of voltage recording with pcNRs.

    table S1. Absorption anisotropy of NRs in the membrane.

    movie S1. Fluorescence movie of pcNR-stained HEK293 cells.

    Reference (56)

  • Supplementary Materials

    This PDF file includes:

    fig. S1. Map of the study area in Kongsfjorden.
    fig. S2. AZFP echograms from ASV.
    fig. S3. EK60 echograms from RV Helmer Hanssen.
    fig. S4. Vertical distribution of zooplankton.
    table S1. Ascent and descent rates of the SSL at 455 kHz in January 2016.