Figure S1.

Characterization of volumetric clustering dynamics and effect of fused protein on construct density, related to Figure 1

(A) Principle of the experiment: 3T3 cells stably expressing GBP-TM were incubated with B(c)-GFP, then A(d)-mCherry to induce rapid clustering.

(B) Cells treated as in (A) were imaged by spinning disk confocal microscopy (left, SDCM) or total internal reflection fluorescence microscopy (right, TIRF). For confocal, images correspond to one optical slice through the ventral surface (top) or XZ slice through the volume of the cell (bottom).

(C) 3T3 cells stably expressing GBP-TM-mScarlet were treated as in (A) with B(c)-GFP, then unlabeled A(d), and GFP-fluorescence was imaged by subcellular light sheet microscopy (see also Video S1). Images correspond to maximum intensity z-projections (MIPs, top and middle), or XZ optical slice (bottom).

(D) Principle of the experiment: 3T3 cells expressing GBP-TM fused to a protein of interest under the control of a doxycycline inducible promoter were treated with doxycycline for the indicated time, before incubation with purified GFP. The GFP signal per cell was measured by flow cytometry as a proxy of the density of the transmembrane construct at the surface of cells expressing the indicated fusion.

(E) GFP intensity per cell expressing the indicated fusion was treated with doxycycline for 3 days and processed as above.

(F) Samples presented in (E) were plotted onto the same graph (3-day samples). Scale bars, 10 μm.