Figure 5.
Vps4 triggers turnover of Vps60-based ESCRT-III polymers. (A) Quantification of fluorescence intensities of indicated subunit and kymographs of time-lapse experiments of addition of Ist1 and Vps4/ATP to SLBs preincubated with Vps60, Vps2, Vps24, Did2, and Ist1 (n ≥ 3, ROI ≥ 30). (B) Quantification of fluorescence intensities of Vps60 and kymographs of time-lapse experiments of the addition of Vps4/ATP and the indicated proteins to SLBs preincubated with Vps60, Vps2, Vps24, Did2, and Ist1 (n ≥ 3, ROI ≥ 20). (C) Quantification of Vps24 fluorescence intensities and kymographs of time-lapse experiments of the addition of Vps4/ATP and Vta1 (when indicated) to SLBs preincubated with Vps60, Vps2, Alexa488-Vps24, Did2, and Ist1 (n = 3, ROI ≥ 18). (D) Quantification of Vps2 fluorescence intensities and kymographs of time-lapse experiments of the addition of Vps4/ATP and Vta1 (when indicated) to SLBs preincubated with Vps60, Vps24, Alexa488-Vps2, Did2, and Ist1 (n = 3, ROI ≥ 35). (E) Quantification of Did2 fluorescence intensities and kymographs of time-lapse experiments of the addition of Vps4/ATP and Vta1 (when indicated) to SLBs preincubated with Vps60, Vps2, Vps24, Alexa488-Did2, and Ist1 (n = 3, ROI ≥ 40). Control experiment without Ist1 together with Vps4 is shown in Fig. S5 A with regard to the data shown here. (F) Quantification of Vps2 intensity and kymographs of time-lapse experiments of addition of Vta1 and Vps4/ATP to SLBs preincubated with Vps2, Vps24, Did1 Ist1, and Snf7 or Vps60 (n = 3, ROI ≥ 80). (G) Cartoon of the model for Vps4-triggered sequential disassembly of Vps60-based ESCRT-III polymer. (H) Confocal images of time-lapse experiments of the addition of Atto647-Vps2, Vps24, and Did2 to SLBs preincubated with Snf7 (green) and Vps60 (red). Single channels are shown in Fig. S5 F. (I) Confocal images of time-lapse experiments of the addition of Atto647-Vps2 and Vps24 to SLBs preincubated with Snf7 (green) and Vps60 (red). Single channels are shown in Fig. S5 G. (J and K) Quantification of Vps2 fluorescence intensities total membrane coverage and non-colocalization with Snf7 patches from experiments described in H and I (n ≥ 3). Scale bar: 10 μm.

Vps4 triggers turnover of Vps60-based ESCRT-III polymers. (A) Quantification of fluorescence intensities of indicated subunit and kymographs of time-lapse experiments of addition of Ist1 and Vps4/ATP to SLBs preincubated with Vps60, Vps2, Vps24, Did2, and Ist1 (n ≥ 3, ROI ≥ 30). (B) Quantification of fluorescence intensities of Vps60 and kymographs of time-lapse experiments of the addition of Vps4/ATP and the indicated proteins to SLBs preincubated with Vps60, Vps2, Vps24, Did2, and Ist1 (n ≥ 3, ROI ≥ 20). (C) Quantification of Vps24 fluorescence intensities and kymographs of time-lapse experiments of the addition of Vps4/ATP and Vta1 (when indicated) to SLBs preincubated with Vps60, Vps2, Alexa488-Vps24, Did2, and Ist1 (n = 3, ROI ≥ 18). (D) Quantification of Vps2 fluorescence intensities and kymographs of time-lapse experiments of the addition of Vps4/ATP and Vta1 (when indicated) to SLBs preincubated with Vps60, Vps24, Alexa488-Vps2, Did2, and Ist1 (n = 3, ROI ≥ 35). (E) Quantification of Did2 fluorescence intensities and kymographs of time-lapse experiments of the addition of Vps4/ATP and Vta1 (when indicated) to SLBs preincubated with Vps60, Vps2, Vps24, Alexa488-Did2, and Ist1 (n = 3, ROI ≥ 40). Control experiment without Ist1 together with Vps4 is shown in Fig. S5 A with regard to the data shown here. (F) Quantification of Vps2 intensity and kymographs of time-lapse experiments of addition of Vta1 and Vps4/ATP to SLBs preincubated with Vps2, Vps24, Did1 Ist1, and Snf7 or Vps60 (n = 3, ROI ≥ 80). (G) Cartoon of the model for Vps4-triggered sequential disassembly of Vps60-based ESCRT-III polymer. (H) Confocal images of time-lapse experiments of the addition of Atto647-Vps2, Vps24, and Did2 to SLBs preincubated with Snf7 (green) and Vps60 (red). Single channels are shown in Fig. S5 F. (I) Confocal images of time-lapse experiments of the addition of Atto647-Vps2 and Vps24 to SLBs preincubated with Snf7 (green) and Vps60 (red). Single channels are shown in Fig. S5 G. (J and K) Quantification of Vps2 fluorescence intensities total membrane coverage and non-colocalization with Snf7 patches from experiments described in H and I (n ≥ 3). Scale bar: 10 μm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal