Figure 2. Nephrin clusters are created via a two-dimensional phase-transition.

(A) Fractional intensity in clusters (blue symbols, left ordinate) and signal variance (red symbols, right ordinate) of p-Nephrin fluorescence on a DOPC bilayer as a function of Nck concentration for 500 nM N-WASP and total p-Nephrin density of ∼2700 molecules/µm². (B) Relative frequency with which a given number of clusters are found within 93 randomly selected 56 × 56 µm regions of a bilayer formed using ∼2500 molecules/µm² Alexa488-labeled p-Nephrin, 1 µM Nck, and 1 µM N-WASP. (C) Size distribution of clusters formed using ∼2500 molecules/µm² Alexa488-labeled p-Nephrin, 1 µM Nck, and 1 µM N-WASP. (D) Puncta formed using 1 µM Nck, 1 µM N-WASP, and low (left) or 4.7-fold higher (right) density of p-Nephrin. Images were autocontrasted for clarity.

DOI: http://dx.doi.org/10.7554/eLife.04123.006

Figure 2—figure supplement 1. Quantitative analysis of the measurement and control of His₈-p-Nephrin density on supported lipid bilayers.

(A) Fluorescence intensity as a function of fluorescent lipid (OG-DHPE) concentration for a solution of small unilamellar vesicles. Fluorescence of liposomes (in solution) containing OG-DHPE were measured at the indicated concentrations of the OG-DHPE concentrations in the x-axis. (B) Fluorescence intensity as a function of p-Nephrin Alexa488 concentrations. Blue points represent data for protein alone, red points represent data for p-Nephrin in the presence of 9.5 μM of Ni²⁺-NTA DOGS. Concentrations of the protein for this standard plot were kept similar to those of the concentration of OG-DHPE in panel A. (C) Fluorescence intensity as a function of OG-DHPE density on supported lipid bilayers. Upper and lower x-axis labels list density as percent total lipid and molecules/µm², respectively. Lines in (A–C) represent a linear fits. (D) Time course of His₈-tagged p-Nephrin Alexa488 dissociation from supported lipid bilayers, monitored by TIRFM, following washes that left 2.8 nM protein in solution above the bilayer. (E) Fluorescence intensity of bilayers containing different percentages of p-Nephrin Alexa488 (with total p-Nephrin density ∼2000 molecules/µm²). The data suggest linearity up to ∼60% labeling.

DOI: http://dx.doi.org/10.7554/eLife.04123.007

Figure 2—figure supplement 2. Quantification of average p-Nephrin density on the bilayer for every titration point shown in Figure 2A.

Y-axis represents p-Nephrin density and x-axis represents the different Nck concentrations of the titration as in Figure 2A. Densities are averages of five different areas of each bilayer. Error bars representing standard deviations are smaller than the symbols.

DOI: http://dx.doi.org/10.7554/eLife.04123.008