Topographical pattern dynamics in passive adhesion of cell membranes - PubMed (original) (raw)

Topographical pattern dynamics in passive adhesion of cell membranes

Alina Hategan et al. Biophys J. 2004 Nov.

Abstract

Strong adhesion of highly active cells often nucleates focal adhesions, synapses, and related structures. Red cells lack such complex adhesion systems and are also nonmotile, but they are shown here to dynamically evolve complex spatial patterns beyond an electrostatic threshold for strong adhesion. Spreading of the cell onto a dense, homogeneous poly-L-lysine surface appears complete in <1 s with occasional blisters that form and dissipate on a similar timescale; distinct rippled or stippled patterns in fluorescently labeled membrane components emerge later, however, on timescales more typical of long-range lipid diffusion (approximately minutes). Within the contact zone, the anionic fluorescent lipid fluorescein phosphoethanolamine is seen to rearrange, forming worm-like rippled or stippled domains of <500 nm that prove independent of whether the cell is intact and sustaining a tension or ruptured. Lipid patterns are accompanied by visible perturbations in Band 3 distribution and weaker perturbations in membrane skeleton actin. Pressing down on the membrane quenches the lipid patterns, revealing a clear topographical basis for pattern formation. Counterion screening and membrane fluctuations likely contribute, but the results primarily highlight the fact that even in adhesion of a passive red cell, regions of strong contact slowly evolve to become interspersed with regions where the membrane is more distant from the surface.

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Figures

FIGURE 1

FIGURE 1

(A) Dynamics of rapid adhesion of a red cell coming into contact with a dense poly-L-lysine substrate as seen by RICM. The fluctuations of the floating cell (first frame and B) are significantly reduced as the cell zips up on the surface (C) and becomes completely attached to the substrate (last frame and D). The adhesion and spreading of the cell is complete in <700 ms. The circular fringes in the RICM image of the attached cell represent reflections off of the upper membrane that become spherical (Hategan et al., 2003). The cap-like bubbles especially obvious in the last two frames exhibit slower dynamics, and arise from reflections off the lower membrane that are otherwise in strong adhesive contact. Scale bar is 5 _μ_m. Intensity profile plots are averages and deviations (Δ) determined from three consecutive frames recorded at 37-ms intervals.

FIGURE 2

FIGURE 2

RICM images of a spread red cell on poly-L-lysine after adhesion (A) and 16 s later (B), showing that some of the bubbles formed in the contact region during adhesion disappear in time. Scale bar is 5 _μ_m.

FIGURE 3

FIGURE 3

Spread red cells already on poly-L-lysine were labeled by addition of Fl-PE, showing that in time (10–15 min) the fluorescent lipid initially in the upper membrane migrates into the adhesive contact region, and eventually yields a fluorescent pattern. (A) Fluorescence image taken immediately after adding the fluorescent lipid to the bulk media of spread red cells shows no cell labeling. (B) After 20 min, many cells show diffuse but uniform fluorescence, which in another 10 min, becomes the network-like pattern (C). The pattern formed appears more clear (D) after the bulk Fl-PE is washed away (removing background fluorescence). Scale bar is 5 _μ_m.

FIGURE 4

FIGURE 4

Fl-PE preincorporated in the red cells is initially uniform while cells spread on poly-L-lysine, but FL-PE later develops into network-like or spotted patterns in the contact region, as shown by TIRF microscopy (A). At slightly higher concentrations of poly-L-lysine, and with a similar number of attached cells, the upper membranes rupture and fuse to form a uniform multicellular membrane surface with dimensions of tens of microns (B). This fused membrane shows the same pattern observed under intact cells, indicating that the pattern formation is independent of the tension in the upper membrane (Hategan et al., 2003), the integrity of the cell, and intracellular components ranging from ATP to hemoglobin. Scale bar is 5 _μ_m.

FIGURE 5

FIGURE 5

Mobility of the fluorescent lipid in the patterned contact region. Photobleaching by closing the iris half of a patterned red cell for 20 s (A) shows that in the bleached region (image B taken immediately after photobleaching) the fluorescent lipids migrate to recover the pattern in ∼1 min (C). Contrast is adjusted in image _C_′ to make the pattern visible, which is shown to remain the same before and after photobleaching. Scale bar is 5 _μ_m.

FIGURE 6

FIGURE 6

The Fl-PE pattern of a red cell is locally quenched in the region where a microrod had been pressed down onto the cell (A_–_B). This provides direct evidence that, when forced into close contact, the Fl-PE can be quenched by poly-L-lysine. The molecular scale view is sketched in C with quenching in the boxed regions. Scale bar is 5 _μ_m.

FIGURE 7

FIGURE 7

Buffer contribution to the pattern in Fl-PE-labeled red cells adhering to a poly-L-lysine surface. In isotonic sucrose buffer, the pattern in the contact region is absent (A), whereas the parallel control sample in PBS shows the pattern (B). From TIRF imaging, intensity variations in PBS are seen to be 50% or less than those in sucrose but not much higher, which implies quenching of fluorescence rather than other mechanisms of intensity gain.

FIGURE 8

FIGURE 8

The adhesion pattern with Fl-PE is dependent on the surface charge density of poly-L-lysine as set by its mass concentration, C, and molecular weight, N. For increasing C and N, weak adhesion with small contact area and an echinocytic (spiky) contour transitions to more uniform spreading and then to patterned spreading. Lytic fusion of the membranes at higher C and N also shows the pattern. At very large C and N, aggregation of the cells before reaching the substrate probably indicates that the poly-L-lysine desorbs from the surface. For the representative images, the indicated phases are separated by linear boundaries of slope −0.65 to −0.88 on this log-log plot. Scale bar is 5 _μ_m.

FIGURE 9

FIGURE 9

(A) The red cell membrane is a complex of lipid and protein, and its components respond differently in the strong adhesion regime. (B) Lipid domain formation is also accompanied by clear spatial perturbations in protein Band 3 distribution, presenting dense and smaller dark point-like regions. This is suggestive of reorganization or quenching of the dye in the regions where the eosin-labeled exoplasmic domain of Band 3 protein is in the favorable nanomolar distance to poly-L-lysine. (C) Rhodamine-labeled actin images of spread red cells suggest that actin is also perturbed, but to a very small degree. (D) Fluorescently labeled poly-L-lysine shows a uniform surface at the same scale. (E) Red cells in strong adhesion on this surface are shown to quench the dye. The AFM scan and height profile also shows the uniformity of hydrated poly-L-lysine on glass. Scale bar is 5 _μ_m.

FIGURE 10

FIGURE 10

Free energy schematic for adhesion and two-stage topographical pattern formation in strong adhesion of the red cell membrane. If membrane tension, γ, exceeds _γ_lysis, the cell ruptures but the pattern persists.

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