Adhesively-tensed cell membranes: lysis kinetics and atomic force microscopy probing - PubMed (original) (raw)

Adhesively-tensed cell membranes: lysis kinetics and atomic force microscopy probing

Alina Hategan et al. Biophys J. 2003 Oct.

Abstract

Membrane tension underlies a range of cell physiological processes. Strong adhesion of the simple red cell is used as a simple model of a spread cell with a finite membrane tension-a state which proves useful for studies of both membrane rupture kinetics and atomic force microscopy (AFM) probing of native structure. In agreement with theories of strong adhesion, the cell takes the form of a spherical cap on a substrate densely coated with poly-L-lysine. The spreading-induced tension, sigma, in the membrane is approximately 1 mN/m, which leads to rupture over many minutes; and sigma is estimated from comparable rupture times in separate micropipette aspiration experiments. Under the sharpened tip of an AFM probe, nano-Newton impingement forces (10-30 nN) are needed to penetrate the tensed erythrocyte membrane, and these forces increase exponentially with tip velocity ( approximately nm/ms). We use the results to clarify how tapping-mode AFM imaging works at high enough tip velocities to avoid rupturing the membrane while progressively compressing it to a approximately 20-nm steric core of lipid and protein. We also demonstrate novel, reproducible AFM imaging of tension-supported membranes in physiological buffer, and we describe a stable, distended network consistent with the spectrin cytoskeleton. Additionally, slow retraction of the AFM tip from the tensed membrane yields tether-extended, multipeak sawtooth patterns of average force approximately 200 pN. In sum we show how adhesive tensioning of the red cell can be used to gain novel insights into native membrane dynamics and structure.

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Figures

FIGURE 1

FIGURE 1

Spherical cap shape of erythrocytes spread in strong adhesion to poly-L-lysine. (A) Reflectance interference microscopy (RIM) image of unfixed, adherent cells in PBS. The concentric fringes are due to the upper membrane since touching it with the AFM tip locally distorts the fringes. (B) AFM contact mode height image of fixed red cells (0.5% glutaraldehyde). Imaging was done at a scan rate of 0.3 Hz. (C) TEM image of rounded rim of adherent red cell after fixation, etching, and thin-sectioning. Although the sections taken were not central slices, the rim's radius of curvature appears to be _R_rim = 125 ± 40 nm (n = four cross-sections). (D) Spherical cap (SC) model calculations of cell height and volume using the spread contact area obtained from both RIM (41 unfixed cells) and tapping-mode AFM (126 unfixed cells) data. Contact mode AFM data on fixed cells (four fixed cells) yields nearly the same values.

FIGURE 2

FIGURE 2

Red cell rupture kinetics. (A) Sketch of cell lysis induced by sustained tension, σ, in the adhering membrane. (B) Bright-field images of spontaneous lysis which shows over tens of minutes that cells in a sealed chamber progressively lyse and lose their internal hemoglobin to become ghosts. (C) Lysis kinetics showing erythrocytes on glass have minimal tendency to lyse whereas the spread erythrocytes lyse with a time constant of 43 min (68 cells were analyzed). (D). Failure time of micropipette-aspirated erythrocyte under tension. The squares are averages of 10–20 cells and are fitted per Evans and Ludwig (2000) (see text). The most relevant data points for the failure times obtained by spreading are also plotted as circles. For the spontaneous lysis time constant determined in C, the fit gives a mean tension in the membrane of σ = 0.8 dynes/cm.

FIGURE 3

FIGURE 3

AFM tip indentation and induced lysis of adhesively-tensed erythrocytes. Sketch (A) and force profile (B) for a tip coming into contact at the center of a spread erythrocyte. The estimated contact point is used to determine the indentation depth to lysis. With increasing velocity, V, of the tip, the lytic force, _f_c, needed to penetrate the tensed erythrocyte membrane increases exponentially as does the indentation depth to the lysis point (C), but the membrane failure events decrease strongly (D). More than 10 cells were tested at each velocity, and the majority of lysis events take place within the first 2–3 indentations. Force curves that do not induce lysis or adhesion to the membrane otherwise prove elastic and reversible.

FIGURE 4

FIGURE 4

Tapping-mode imaging of spread and unfixed erythrocytes. (A) Sketch of an adhesively-tensed erythrocyte indented down during imaging from a maximum height of ∼2.5 _μ_m to an average compressed height of 42 nm. (B) Tapping-mode amplitude image of spread and unfixed erythrocytes demonstrates compression of the spherical cap shape. Scale bar is 5 _μ_m and scan rate is 0.3 Hz (velocity of tip 18 _μ_m/s). (C) Within 1–2 _μ_m of the cell margin, the average height is 42 ± 15 nm (±SD). Overlapping cells show twice this average height.

FIGURE 5

FIGURE 5

Sketch representing the extension and dilation of the cytoskeleton in an intact red cell during either AFM indentation (A) or micropipette aspiration (B) (Discher et al., 1994).

FIGURE 6

FIGURE 6

Membrane tension and the immobilized cap shape allow highly stable and reproducible tapping-mode imaging (height images) of the unfixed red cell membrane (A, B). For these, scan areas are close to the center and are just 1 _μ_m2 in area. In contrast, unstable images are collected from a semiadherent echinocyte (C, D) which is not tensed. All of the detail images of the external face of the membrane show corrugated networklike structures ∼7 nm in height and suggest indentation of the membrane down to the cytoskeletal network. When tensed, apparent distension of spectrin molecules in the network show lengths consistent with ∼200-nm contours in an extended state (A, B). Scan rates are 2 Hz (A, B) and 0.5 Hz (C, D).

FIGURE 7

FIGURE 7

AFM imaging of the unfixed cytoplasmic face of a lysed red cell shows 3–4-nm high features for the cytoskeletal meshwork on the adherent membrane. This is in good agreement with Swihart et al. (2001). The scan rate is 1 Hz.

FIGURE 8

FIGURE 8

In the retraction phase of the indentation curves, the tensed membrane often adheres to the AFM tip (∼50% frequency) and deforms upward (A), suggestive of cusp and tether formation. Continued pulling frequently showed multipeak sawtooth patterns extending over several hundred nm (B, C), with a major force peak averaging 200 pN.

FIGURE A1

FIGURE A1

Small force indentation behavior. (A) Schematic for indenting a prestressed membrane. (B) Indentation curve showing the hard limit as a dashed line at 1800 nm, and (C) low force behavior for three cells using the same tip (10 pN/nm). At the inflection with the baseline where membrane contact first appears, a line is fit with a slope of 0.2 ± 0.1 pN/nm (four cells).

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