Distribution of GFP-Nrx in a HEK293 cell in contact with GPI-Nlg–supported lipid bilayer. (a,b) Confocal microscopy images of GFP-Nrx at the cell-bilayer contact extracted from the z series for a supported POPC bilayer without (a) and with GPI-Nlg (b). Scale bar, 5 μm. The rainbow scale was established such that red corresponds to the maximum GFP fluorescence intensity at the equatorial plane, where the protein concentration is maximal, and blue corresponds to the background fluorescence. The red circle outlines the cell contour at the equatorial plane. Schematic representations of GFP-Nrx distribution and the bound cell shape are shown next to each condition. z₀ marks the plane imaged. (c) Co-localization of GPI-Nlg in the supported lipid bilayer with GFP-Nrx expressed in HEK293 cells. Left, distribution of GFP-Nrx in the cell in the optical slice contacting the supported lipid bilayer containing GPI-Nlg. Right, distribution of GPI-Nlg labeled with Alexa-555 in the supported lipid bilayers. Bottom, fluorescence intensity profiles measured along the dash line for Alexa-555–GPI-Nlg (red) and GFP-Nrx (green). In the absence of cells, the fluorescence profile of Alexa-555–GPI-Nlg bilayers fluctuates around the background level (gray).

Correlation between distribution of GFP-Nrx in cells and cell immobilization. (a) Histogram of the GFP-Nrx fluorescence intensity (I_GFP-Nrx) at z₀, the position of the cell-bilayer interface for plain supported lipid bilayers (black), bilayers containing GPI-Nlg (red) and bilayers containing GPI-Nlg(K578A/V579A) (green). I_GFP-Nrx(z₀) is normalized by the total GFP-Nrx for the cell (I_{GFP-Nrx cell total}). The values were binned in 0.2 intervals. The mean of the normalized GFP-Nrx fluorescence at the contact plain is: 0.31 (s.e.m. = 0.06, n = 11) for cells on plain bilayer; 0.53 (s.e.m. = 0.05, n = 20) for cells on GPI-Nlg–containing bilayers and 0.27 (s.e.m. = 0.03, n = 26) for cells on GPI-Nlg(K578A/V579A)–containing bilayers. (b) z-axis profiles of GFP-Nrx distribution throughout a HEK293 cell. The horizontal axis represents GFP-Nrx fluorescence at one z position (I_GFP-Nrx(z)) normalized by I_{GFP-Nrx cell total}. The vertical axis represents the z-axis position normalized by the cell height. This profile reveals a specific skew of distribution to the GPI-Nlg bilayer (red triangles), whereas the profiles for plain POPC bilayers (black lines) and GPI-Nlg(K578A/V579A) (green circles) are more symmetric about the cell equatorial plane. (c) Displacement range for GFP-Nrx cells on supported lipid bilayers, containing from left to right, GPI-Nlg, GPI-Nlg(K578A/V579A), POPC only, GPI-AP. The color code is as for Figure 3c,d. (d) Bar graph of the ratio between the contact region, A_contact, and the equatorial area, A_equatorial, for cells on supported lipid bilayer containing GPI-Nlg (red), GPI-Nlg(K578A/V579A) (green) and plain POPC (black).

Reconstitution of GPI-Nlg in a fluid-supported lipid bilayer. (a) 100-nm SUVs incorporated with Alexa-555–labeled GPI-Nlg form a supported lipid bilayer by fusion with an etched clean glass surface. (b) Alexa-555–GPI-Nlg protein diffusing in the supported lipid bilayer imaged by TIRF for 3 s at 80 frames per second. Each bright spot corresponds to a single protein. (c) Corresponding single-molecule trajectories. Scale bars, 5 μm. (d) Averaged mean squared displacement calculated for all trajectories. For time intervals <1 s, the mean squared displacement is a linear function of time as expected for two-dimensional thermal diffusion. A linear fit to the mean squared displacement led to a translational diffusion coefficient of 1.10 ± 0.01 μm² s⁻¹ for the GPI-Nlg protein. Irreversible photobleaching of the fluorophores shorten the fluorescent tracks, leaving fewer than ten trajectories for time intervals >1 s, which increases the uncertainty on the mean squared displacement for t > 1 s.

Cell immobilization associated with formation of Nrx-Nlg puncta. (a) Bright-field time series of three cells in contact with a GPI-Nlg lipid bilayer. The white dot marks the position of the only non-expressing cell that drifts along the bilayer. Its trajectory is marked by magenta lines. (b) Corresponding confocal microscopy images of GFP-Nrx at the cell-bilayer contact. The upper cell (dashed circle) has several GFP-Nrx puncta and is fixed in place, whereas the lower cell has a diffuse GFP-Nrx distribution and rotates about its center, as marked by the arrow. Scale bar, 10 μm. (c) Typical trajectories observed for GFP-Nrx cells dropped onto a plain POPC supported lipid bilayer. Scale bar, 10 μm. The bar graphs represent all the measured displacement for cells dropped onto plain POPC lipid bilayers color-coded according the displacement dl per minute: blue for dl< R/10, green for R/10 o dl < R/3, orange for R/3 < dl < R and red for dl>R. (d) Range of displacements observed for GFP-Nrx cells dropped onto GPI-Nlg lipid bilayer. The bar graphs represent all the measured displacement for cells dropped onto GPI-Nlg lipid bilayers.

Inversion assay: relation between Nrx-Nlg distribution and adhesion strength. (a) Schematic of the observation chamber after cells are dropped onto the supported lipid bilayer (left) and after the chamber is inverted so that the cells are pulled from the bilayer by gravity (right). (b–e) Overlay images of the bright field, showing all cells, and GFP fluorescence images, showing GFP-Nrx–expressing cells, on a 737 × 737–μm supported lipid bilayer. Shown are cells on a plain lipid bilayer before (b) and 5 min after inversion (d), and cells seeded on a GPI-Nlg lipid bilayer before (c) and 5 min after inversion (e). Cells only adhere to the GPI-Nlg lipid bilayer. The bright stripes in c are artifacts caused by light diffraction. (f) Bar graph of number of cells seeded onto lipid bilayer containing: GPI-Nlg lipid bilayer (red) or GPI-Nlg(K578A/V579A) (green), and number adhering after chamber inversion for control cells and for cells expressing GFP-Nrx. Values are mean ± s.e.m. of five sets of samples. Note that the y-axis is broken to facilitate comparison.