Fig. 4. ESCRT membrane scission is mediated by tube constriction.

(A and B) Force profiles of the same events shown in Fig. 3 were normalized to their respective baseline tube holding force f₀. ATP released by UV uncaging leads to a rise in force and eventually scission (indicated by the sharp drop of force at the end of the trace, 2 of 17). (C and D) Simultaneous observation of the tube radius revealed a continuous radius decrease. Force and radius plots are overlaid with predictions from the Helfrich model for a tube of unchanged bending rigidity under constant tension (dashed orange lines, see Methods). (E and F) Normalized and bleaching-corrected fluorescence intensity profiles of the scission events for the tube in the three fluorescence channels (Snf7, green; Vps4, cyan; membrane, red; ± SD shown as shaded area). After ATP uncaging, the tube membrane fluorescence intensity decreased while the tube Snf7 and Vps4 intensities increased. (G and H) The GUV membrane fluorescence intensity profile over the time course of the scission event was essentially constant and showed no correlation with the force (Snf7, green; Vps4, cyan; membrane, red; ± SD shown as shaded area). (I) Fluorescence intensity profiles of the GUV membrane at the start and at the end of the scission trace. No increase in protein fluorescence on the membrane was detected. (J) GUV aspiration projection at the beginning, middle and end of the scission trace, showing essentially no change. (K to N) Averages (N = 17 ± SD) of force, radius and tube fluorescence show consistent force rise, radius decrease and fluorescence increase in the tube. (O) Correlation (r) between experiment and prediction for radius and force, N=17.