Extended Data Fig. 6: Membrane-tethered RNA recruits Whi3 and forms condensates.

(a) Confocal slices of ER and CLN3 smFISH in Ashbya cells. Arrows indicate example CLN3 puncta showing ER co-localization. (b) Images of CLN3 puncta tethered to membrane (left) or PEG surface (right) prior to addition of Whi3. Images contrasted equally. (c) Initial densities of CLN3 puncta on membranes or PEG surfaces prior to addition of Whi3. Black bars show mean ± first s.d., n = 90 and 60 images from 18 and 12 independent membrane and PEG experiments, respectively. (d) Average CLN3 puncta intensity on membrane or PEG surfaces as a function of time after addition of 50 nM Whi3. PEG-tethered CLN3 did not recruit Whi3 or cluster to the same extent as membrane-tethered CLN3, despite the higher initial CLN3 density on PEG surfaces. Data are mean ± 95% c.i. n = 1,059–1,335 and 1,804–1,987 puncta per data point in membrane and PEG experiments, respectively, from 3 biologically independent samples in each experiment. Exact n per data point provided in Source Data Extended Data Fig. 6d. (e) Histograms of scaled Whi3 puncta intensity from Ashbya cells and membrane-tethered CLN3 experiments after clustering by 50 nM Whi3. Scaled distributions obtained by dividing by the distribution means. n = 13,198 and 867 Whi3 puncta in membrane-tethered CLN3 experiment and in live cells, respectively. (f) Membrane-tethered RNA condensates 15 min after exposure to Whi3 at the indicated concentrations. CLN3 and Whi3 channels contrasted equally in all images. (g-h) Intensity of CLN3 (g) and Whi3 (h) within membrane-tethered RNA condensates as a function of bulk Whi3 concentration. Data are mean ± 95% c.i. in (g-h). Each data point in (g-h) contains n = 7,484–35,945 and 9,238–37,992 puncta in membrane and PEG experiments, respectively, from 3 biologically independent samples in each experiment. Exact n per data point provided in Source Data Extended Data Fig. 6g,h. SLB membrane composition: 99 mol% DOPC, 1 mol% DOPE cap-biotin.

Source data