Figure 2. Single-molecule coincidence and ALPHAScreen mapping of interactions among cavin1, 2 and 3.

(A) Histogram of single-molecule coincidence between cavin1-GFP and cavin2-Cherry co-transfected in MCF-7 cells. The coincidence is calculated as the ratio of intensity in the Cherry channel divided by the sum of the signals in the GFP and Cherry channels. GFP-only bursts show a coincidence at 0 and Cherry-only oligomers are located at coincidence = 1. For the oligomers containing both fluorophores, the coincidence ratio is a measure of the stoichiometry of the assembly. (B) Same as (A), coincidence between cavin1-GFP and cavin3-Cherry. (C) Same as (A), coincidence between cavin2-GFP and cavin3-Cherry. (D–F): using cell-free protein expression, three-dimensional histograms of single-molecule coincidence between GFP and Cherry cavin proteins at expression ratios spaning from 100% GFP to 100% Cherry. For each DNA ratio of cavin-GFP and cavin-Cherry, we collected >1000 bursts and plotted the corresponding histograms of coincidence. We varied the ratio of cavins and created a stack of 10 histograms representing the various stoichiometries. The histograms for individual pairs were then aggregated into 3D plots. (D) Evolution of mixed oligomers of cavin1-GFP and cavin2-Cherry revealing formation of oligomers with a full range of stoichiometries. (E) The cavin1 and cavin3 plot shows formation of cavin1-cavin3 oligomers with predominantly 3/1 composition. (F) The cavin2 and cavin3 plot shows that these proteins do not form mixed oligomers. (G) Schematic representation of ALPHAScreen principle. This bead–bead assay relies on transfer of singlet oxygen from a donor bead to a luminescent acceptor bead when protein–protein interactions bring the beads within 200 nm (see ‘Materials and methods’ and SI for details). (H) A plot of ALPHAScreen signal across the concentrations of cavin proteins attached to donor and acceptor beads. The interactions for cavin2 and cavin3 in the presence or absence of cavin1 display amplitudes close to the background signal. (I) Values obtained for cavin1-GFP and cavin2-myc, cavin1-GFP and cavin3-myc, cavin2-GFP and cavin2-myc reveal robust interactions. However the curves obtained for cavin3-GFP and cavin2-myc, cavin2-GFP and cavin3-myc demonstrate that cavin2 and cavin3 cannot bind to each other. The triple co-expression of cavin2-GFP, cavin3-myc and untagged cavin1 results in no change in binding, suggesting that cavin1 cannot act as a bridge between cavin2 and cavin3.

DOI: http://dx.doi.org/10.7554/eLife.01434.004

Figure 2—figure supplement 1. Single-molecule fluorescence trace of cavin1-GFP during expression in the cell-free system.

The fluorescence trace shows the real-time oligomerization of cavin1, occurring within 15 min. While all the GFP fluorophores are not yet folded and fluorescent, we can already detect bursts of large amplitude suggesting the formation of oligomers at very low concentration. We estimate that at 15 min, the concentration in the cell-free expression system has not reached 10 nM. We stopped translation after 15 min by diluting the sample and performed single-molecule analysis; the residence times and brightness obtained correspond to a population of oligomerized cavin1.

DOI: http://dx.doi.org/10.7554/eLife.01434.005

Figure 2—figure supplement 2. Comparison of cavin1 oligomers observed in MCF-7 cells and expressed in the cell-free system.

(A) Plot of the residence times as a function of number of experiments for cavin1-GFP in MCF-7 cells. (B) Histogram of sizes measured by single-molecule diffusion and calibrated by the diffusion of GFP, as shown in Figure 1 of the main text. (C) Plot of the residence times as a function of number of experiments for cavin1-GFP expressed in the cell-free system. (D) Histogram of apparent sizes of cavin1-GFP expressed in cell-free system. The distributions of residence times and apparent sizes obtained from cell extract and in the cell-free expression system match closely, suggesting that cavin1 has an intrinsic propensity to form oligomers.

DOI: http://dx.doi.org/10.7554/eLife.01434.006

Figure 2—figure supplement 3. Principle of the ALPHA screen.

(top panel) The donor bead is coated with protein A. A laser pulse at 680 nm triggers the release of singlet oxygen from the donor bead. The singlet oxygen has a half-life of 4 μs and can diffuse over 200 nm. If an interaction occurs between protein A and protein B, the acceptor bead is brought into proximity of the donor bead. The singlet oxygen will react with thioxene derivatives encapsulated in the acceptor bead, resulting in luminescence, emitted between 520 and 620 nm. (bottom panel) The ‘hook’ effect is a signature of the Screen signal. This effect appears due to loading effects on the beads. If the concentration of interacting proteins is too low, the beads do not bind strongly and the luminescence signal is low. At optimal protein concentration, bead–bead contacts are maximized as all the beads are covered with proteins. When the system is overloaded with proteins, unbound proteins are competing with bead-immobilized proteins, diminishing the number of bead–bead interactions and lowering the AlphaScreen signal.

DOI: http://dx.doi.org/10.7554/eLife.01434.007

Figure 2—figure supplement 4. Pull-down analysis of the cavin complex formation in MCF-7 cells.

MCF-7 cells were transfected with equal amounts of GFP and Cherry alone, GFP alone + Cavin 1-Cherry, GFP + Cavin 2-Cherry, GFP + Cavin 3-Cherry, Cavin 1-GFP and Cavin 2-Cherry, Cavin 1-GFP and Cavin 3-Cherry, Cavin 2-GFP and Cavin 3-Cherry and Cavin 1-Flag with Cavin 3-GFP and Cavin 2-Cherry. Post-nuclear supernatant (soluble cytoplasmic fraction) were prepared by extensive washing of cells in PBS. Cells were then scraped into PBS containing protease and phosphatase inhibitors followed by mechanic disruption by syringe lysis. Cells were then pelleted at 2000 rpm, 10 min at 4°C. 120th of the supernatant was retained as the starting material (B). The remaining supernatant was mixed with 20 μl of prewashed GFP Trap beads for 30 min at 4°C on a rotating wheel. The beads were pelleted and washed three times in PBS supplemented with protease and phosphatase inhibitors and were boiled in 4X Sample buffer for 75°C for 2.5 min to preserve the fluorescence. Sample were separated by SDS-PAGE and the fluorescence corresponding to each of the overexpressed GFP or Cherry tagged Cavin construct was detected and quantification in the gel using the BioRad ChemiDoc MP Imaging System.

DOI: http://dx.doi.org/10.7554/eLife.01434.008