Studying the Nucleated Mammalian Cell Membrane by Single Molecule Approaches

<div><p>The cell membrane plays a key role in compartmentalization, nutrient transportation and signal transduction, while the pattern of protein distribution at both cytoplasmic and ectoplasmic sides of the cell membrane remains elusive. Using a combination of single-molecule techniques, including atomic force microscopy (AFM), single molecule force spectroscopy (SMFS) and stochastic optical reconstruction microscopy (STORM), to study the structure of nucleated cell membranes, we found that (1) proteins at the ectoplasmic side of the cell membrane form a dense protein layer (4 nm) on top of a lipid bilayer; (2) proteins aggregate to form islands evenly dispersed at the cytoplasmic side of the cell membrane with a height of about 10–12 nm; (3) cholesterol-enriched domains exist within the cell membrane; (4) carbohydrates stay in microdomains at the ectoplasmic side; and (5) exposed amino groups are asymmetrically distributed on both sides. Based on these observations, we proposed a Protein Layer-Lipid-Protein Island (PLLPI) model, to provide a better understanding of cell membrane structure, membrane trafficking and viral fusion mechanisms.</p></div

The proposed Protein Layer-Lipid-Protein Island (PLLPI) model of the cell membrane.

<p>(A and B) The top and bottom view of the cell membrane, respectively. The proteins on the ectoplasmic side of the cell membrane form a dense protein layer to show a smooth feature (A); the proteins on the cytoplasmic side tend to form dispersed microdomains (B). (C) The size of the cell membrane. The total height of the cell membrane is 20 nm, which is composed of the ectoplasmic protein layer (4 nm), lipid bilayer (4 nm) and cytoplasmic protein layer (12 nm).</p

Detecting the amino groups on both sides of the cell membrane by single- molecule force spectroscopy.

<p>(A) The scheme of AFM tip functionalized with aldehyde group. (B and C) The typical force curves acquired on the cytoplasmic and ectoplasmic side of the cell membrane, respectively. The black and red lines represent the approaching and withdrawn curves, respectively.</p

Membrane height after treatment with enzyme and solvent.

<p>EP, ectoplasmic side of membrane; CP, cytoplasmic side of membrane; D_enzyme, depth of the pits after enzyme digestion; D_MβCD/D_TX, depth of the pits after treatment with MβCD or TX (Triton X-100); H_enzyme, height of the membrane after digestion by enzyme; H_MβCD/H_TX, height of the membrane after treatment with MβCD or TX; Unit: nm.</p

The cytoplasmic side of the cell membrane treated with trypsin or proteinase K.

<p>(A) AFM image of the cytoplasmic side of the cell membrane after digestion with trypsin for 1 h. The single and double layers of membranes are indicated by green and pink arrows, respectively. (B) The membranes were treated <i>in situ</i> with 0.1% Triton X-100. (C) and (E) The magnified images from (A) and (B), respectively. (D) and (F) The cross section analysis along the green lines in (C) and (E), respectively. (G) and (H) The images of the cytoplasmic side of membranes after treatment with proteinase K and MβCD in sequence. (I) The magnified image of the green square area in (H). (J) and (K) The size and depth distributions of the pits eroded with MβCD in (H and I), respectively. (L) and (M) The STORM images of band 3 labeled with anti-band 3-Cy5 on the cytoplasmic side of the cell membrane before and after treatment with MβCD, respectively. Scale bars: 2 µm in (A–B), 200 nm in (C–E), 1 µm in (G–H), 300 nm in (I), and 4 µm in (L–M).</p

Imaging the ectoplasmic side of the cell membranes from various types of mammalian cells.

<p>(A) The scheme of this work. Cells were cultured on cover slips (A1), (A2) and (A3). The ectoplasmic and cytoplasmic sides of membranes were prepared separately and then investigated with AFM imaging, single-molecule force spectroscopy (SMFS), and STORM, respectively. (B) The ectoplasmic side of MDCK cell membrane was directly imaged on a living cell. (C) The image of the ectoplasmic side of the cell membrane (MDCK cells) prepared by shearing open the cells on a cover slip. (D) The image of the ectoplasmic side of the cell membrane (MDCK cells) prepared by centrifugation. (E and F) The ectoplasmic side of A549 (E) and HeLa (F) cell membranes prepared by the shearing open approach, respectively. Scale bars: 100 nm in (B–F).</p

Imaging the cytoplasmic side of the cell membrane.

<p>(A) The scheme for preparing the cytoplasmic side of the cell membrane. (B and C) The fluorescent images of the cytoplasmic side of the cell membrane before and after incubation with high-salt buffer, respectively. The red membrane patches represent the lipid bilayer labeled with DiI, and the green fibers represent the actin filaments labeled with phalloidin-FITC. (D and E) The AFM topographic images of the cytoplasmic side of the cell membrane before and after incubation with high-salt buffer, respectively. (F) The high magnification image of the cytoplasmic side of the cell membrane. (G–I) Cross section analysis along the green line in (D–F), respectively. Scale bars: 7 µm in (B)–(E); 500 nm in (F).</p

Digestion of the ectoplasmic side of the cell membrane with proteinase K or collagenase 3.

<p>(A) The AFM topographic image of the ectoplasmic side of the cell membrane. (B and C) The AFM topographic image of the ectoplasmic side of cell membranes treated with proteinase K (B) and MβCD (C) in sequence. (D, F and H) The magnified images from (A, B and C), respectively, showing the gradual deepening of the pits. (E, G and I) The cross section analysis along the green lines in (D, F and H), respectively. (J and K) The depth and width distributions of the pits after proteinase K treatment, respectively. (L and M) The depth and width distributions of the pits after MβCD treatment, respectively. (N) The AFM topographic image of the ectoplasmic side of the cell membrane without treatment. (O and P) The AFM topographic image of the ectoplasmic side of the cell membrane treated <i>in situ</i> with collagenase 3 and MβCD in sequence, respectively. (Q, R and S) The AFM amplitude images corresponding to (N, O and P), respectively. (T, V and X) The magnified images of (N, O and P), respectively. (U, W and Y) The cross section analysis along the green lines in (T, V and X), respectively. Scale bars: 300 nm in (A–C), 80 nm in (D, F and H), 300 nm in (N–S), and 50 nm in (T, V and X).</p

Detecting the domains of carbohydrates on the ectoplasmic surface by STORM.

<p>(A) The principle of STORM. (B) The STORM image of mannose clusters labeled with MNA-Cy5 on the ectoplasmic surface of the cell membranes. (C) The size distribution of the mannose clusters. Scale bar: 8 µm in (B).</p