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Relaxation of Loaded ESCRT-III Spiral Springs Drives Membrane Deformation.

Chiaruttini N, Redondo-Morata L, Colom A, Humbert F, Lenz M, Scheuring S, Roux A - Cell (2015)

Bottom Line: We reasoned that Snf7 spirals could function as spiral springs.Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature.This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission.

View Article: PubMed Central - PubMed

Affiliation: University of Geneva, Department of Biochemistry, quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland.

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Nucleation and Disruption of Snf7, Related to Figure 4All images are acquired with TIRF Microcopy. (A) Control of Snf7 polymerization induced by ESCRT-II and Vps20 proteins. Kymograph of a membrane slice over time. Fluorescents dots appear as lines when Snf7 nuclei are polymerized. Lines appear only when both Vps20 and ESCRT-II are present (both kymograph, lower panel). Snf7 and Vps20 without ESCRT-II (left kymograph middle panel) as well as Snf7 and ESCRT-II without Vps20 (right kymograph middle panel) do not nucleate Snf7 filaments. (B) Photobleaching induces breaks in Snf7 dotted structures. Snf7 dots are formed by incubation of Snf7 at 300 nM for 1 hr on DOPC 60% /DOPS 40% membrane. Snf7 is then washed out. Snf7 dots are subsequently imaged at a high frequency (1 fps) and under long exposure time and high laser power. When an antibleaching solution (AB) is present in the chamber, the dots remain intact (first and second image). However 5 min and 15 min after the antibleaching solution is washed out (third and fourth image), dots are disrupted into wider structures showing multiple maxima (see 2 × 2 μm insets for details). Structures located outside the imaged field of view are not affected by the removal of the antibleaching solution. (C) Photobleaching triggers patch formation from Snf7 dots. Compared to (B), Snf7 is maintained in the chamber at 300 nM throughout the experiment. In this case, the removal of the antibleaching solution (Snf7 - AB point of the kymograph) synchronously triggers the transition from Snf7 dots to patches. (D) Histogram of the Snf7 filament radius distribution after nanodissection (total of three experiments, see Figure 4D).
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figs4: Nucleation and Disruption of Snf7, Related to Figure 4All images are acquired with TIRF Microcopy. (A) Control of Snf7 polymerization induced by ESCRT-II and Vps20 proteins. Kymograph of a membrane slice over time. Fluorescents dots appear as lines when Snf7 nuclei are polymerized. Lines appear only when both Vps20 and ESCRT-II are present (both kymograph, lower panel). Snf7 and Vps20 without ESCRT-II (left kymograph middle panel) as well as Snf7 and ESCRT-II without Vps20 (right kymograph middle panel) do not nucleate Snf7 filaments. (B) Photobleaching induces breaks in Snf7 dotted structures. Snf7 dots are formed by incubation of Snf7 at 300 nM for 1 hr on DOPC 60% /DOPS 40% membrane. Snf7 is then washed out. Snf7 dots are subsequently imaged at a high frequency (1 fps) and under long exposure time and high laser power. When an antibleaching solution (AB) is present in the chamber, the dots remain intact (first and second image). However 5 min and 15 min after the antibleaching solution is washed out (third and fourth image), dots are disrupted into wider structures showing multiple maxima (see 2 × 2 μm insets for details). Structures located outside the imaged field of view are not affected by the removal of the antibleaching solution. (C) Photobleaching triggers patch formation from Snf7 dots. Compared to (B), Snf7 is maintained in the chamber at 300 nM throughout the experiment. In this case, the removal of the antibleaching solution (Snf7 - AB point of the kymograph) synchronously triggers the transition from Snf7 dots to patches. (D) Histogram of the Snf7 filament radius distribution after nanodissection (total of three experiments, see Figure 4D).

Mentions: To characterize the initial events leading to patch formation, we first studied the early steps of Snf7 patch nucleation by TIRFM, which allowed us to quantify the approximate number of Snf7 molecules in diffraction limited spots from their fluorescence intensity (see Experimental Procedures for quantification). Patch nucleation started with the appearance of a fluorescent diffraction limited spot (nucleus, Figure 4A) containing 50 ± 20 monomers (n = 9). At a Snf7 concentration of 300 nM, the intensity of the nuclei remained constant for several minutes until these nuclei started to grow (Figure 4A). Under these conditions, the number of nuclei is very low (Figure 1C). To increase the number of nuclei and to obtain more robust statistics, we nucleated Snf7 assemblies by adding 1 μM ESCRT-II and 1 μM Vps20 to a 75 nM Snf7 solution. Under these conditions, many Snf7 nuclei appeared on the membrane surface (Figure 4B and Figure S4A) and remained stable for several tens of minutes, consistent with our observations with Snf7 alone (see Figure 4A).


Relaxation of Loaded ESCRT-III Spiral Springs Drives Membrane Deformation.

Chiaruttini N, Redondo-Morata L, Colom A, Humbert F, Lenz M, Scheuring S, Roux A - Cell (2015)

Nucleation and Disruption of Snf7, Related to Figure 4All images are acquired with TIRF Microcopy. (A) Control of Snf7 polymerization induced by ESCRT-II and Vps20 proteins. Kymograph of a membrane slice over time. Fluorescents dots appear as lines when Snf7 nuclei are polymerized. Lines appear only when both Vps20 and ESCRT-II are present (both kymograph, lower panel). Snf7 and Vps20 without ESCRT-II (left kymograph middle panel) as well as Snf7 and ESCRT-II without Vps20 (right kymograph middle panel) do not nucleate Snf7 filaments. (B) Photobleaching induces breaks in Snf7 dotted structures. Snf7 dots are formed by incubation of Snf7 at 300 nM for 1 hr on DOPC 60% /DOPS 40% membrane. Snf7 is then washed out. Snf7 dots are subsequently imaged at a high frequency (1 fps) and under long exposure time and high laser power. When an antibleaching solution (AB) is present in the chamber, the dots remain intact (first and second image). However 5 min and 15 min after the antibleaching solution is washed out (third and fourth image), dots are disrupted into wider structures showing multiple maxima (see 2 × 2 μm insets for details). Structures located outside the imaged field of view are not affected by the removal of the antibleaching solution. (C) Photobleaching triggers patch formation from Snf7 dots. Compared to (B), Snf7 is maintained in the chamber at 300 nM throughout the experiment. In this case, the removal of the antibleaching solution (Snf7 - AB point of the kymograph) synchronously triggers the transition from Snf7 dots to patches. (D) Histogram of the Snf7 filament radius distribution after nanodissection (total of three experiments, see Figure 4D).
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figs4: Nucleation and Disruption of Snf7, Related to Figure 4All images are acquired with TIRF Microcopy. (A) Control of Snf7 polymerization induced by ESCRT-II and Vps20 proteins. Kymograph of a membrane slice over time. Fluorescents dots appear as lines when Snf7 nuclei are polymerized. Lines appear only when both Vps20 and ESCRT-II are present (both kymograph, lower panel). Snf7 and Vps20 without ESCRT-II (left kymograph middle panel) as well as Snf7 and ESCRT-II without Vps20 (right kymograph middle panel) do not nucleate Snf7 filaments. (B) Photobleaching induces breaks in Snf7 dotted structures. Snf7 dots are formed by incubation of Snf7 at 300 nM for 1 hr on DOPC 60% /DOPS 40% membrane. Snf7 is then washed out. Snf7 dots are subsequently imaged at a high frequency (1 fps) and under long exposure time and high laser power. When an antibleaching solution (AB) is present in the chamber, the dots remain intact (first and second image). However 5 min and 15 min after the antibleaching solution is washed out (third and fourth image), dots are disrupted into wider structures showing multiple maxima (see 2 × 2 μm insets for details). Structures located outside the imaged field of view are not affected by the removal of the antibleaching solution. (C) Photobleaching triggers patch formation from Snf7 dots. Compared to (B), Snf7 is maintained in the chamber at 300 nM throughout the experiment. In this case, the removal of the antibleaching solution (Snf7 - AB point of the kymograph) synchronously triggers the transition from Snf7 dots to patches. (D) Histogram of the Snf7 filament radius distribution after nanodissection (total of three experiments, see Figure 4D).
Mentions: To characterize the initial events leading to patch formation, we first studied the early steps of Snf7 patch nucleation by TIRFM, which allowed us to quantify the approximate number of Snf7 molecules in diffraction limited spots from their fluorescence intensity (see Experimental Procedures for quantification). Patch nucleation started with the appearance of a fluorescent diffraction limited spot (nucleus, Figure 4A) containing 50 ± 20 monomers (n = 9). At a Snf7 concentration of 300 nM, the intensity of the nuclei remained constant for several minutes until these nuclei started to grow (Figure 4A). Under these conditions, the number of nuclei is very low (Figure 1C). To increase the number of nuclei and to obtain more robust statistics, we nucleated Snf7 assemblies by adding 1 μM ESCRT-II and 1 μM Vps20 to a 75 nM Snf7 solution. Under these conditions, many Snf7 nuclei appeared on the membrane surface (Figure 4B and Figure S4A) and remained stable for several tens of minutes, consistent with our observations with Snf7 alone (see Figure 4A).

Bottom Line: We reasoned that Snf7 spirals could function as spiral springs.Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature.This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission.

View Article: PubMed Central - PubMed

Affiliation: University of Geneva, Department of Biochemistry, quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland.

Show MeSH
Related in: MedlinePlus