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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 Growth of Snf7 Spirals on Supported Membranes(A) Left: TIRF microscopy kymographs of the nucleation of single Snf7 patches (green) at [Snf7] = 300 nM. Arrows indicate single ring to multiple spirals transition as postulated from the interpretation of these observations (right).(B) TIRF microscopy image of Snf7-Alexa488 dots (green) nucleated by ESCRT-II, [Snf7] = 75 nM, [Vps20] = 1 μM, [ESCRT-II] = 1 μM. Inset: zoom on 4 diffraction-limited spots (scale bar, 2 μm).(C) Histogram of the estimated number of Snf7 molecules within the dots nucleated by ESCRT-II (n = 1856).(D) HS-AFM nanodissection experiment (see text) of Snf7 spirals. 2 cycles of high AFM force were applied, between 0 s and 10 s, and between 10 s and 20 s.(E) HS-AFM time-lapse sequence showing the apparition of a new Snf7 spiral from pre-existing ones. Arrowheads show: filament protruding from a spiral (t = 8.5 s), filament curling from its tip (t = 17.0 s), and forming a small spiral (t = 37.4 s), growth of a second turn in the spiral (t = 152.2 s) and filament rearrangements (t = 164.9 s).(F) HS-AFM time-lapse sequence of an isolated Snf7 spiral. Arrowheads show: growth of the spiral at the two-turn stage (t = 67.2 s), and filament split (t = 75.7 s) leading to the three turns stage.(G) The equatorial kymograph (yellow rectangle) of this growing spiral: the innermost turn radius decreases from 22 nm to 14 nm upon formation of the third turn.(H) Dynamics of filament splitting and fusing in two Snf7 spirals (rows) observed by HS-AFM. Arrowheads show displacement of the splitting points.(I) Time plot of the outer radius of five growing Snf7 spirals followed by HS-AFM. The origin of all curves is the apparition of the first turn. The thick curve is the average of all curves. [Snf7] = 1 μM. See also Figure S4.
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fig4: Nucleation and Growth of Snf7 Spirals on Supported Membranes(A) Left: TIRF microscopy kymographs of the nucleation of single Snf7 patches (green) at [Snf7] = 300 nM. Arrows indicate single ring to multiple spirals transition as postulated from the interpretation of these observations (right).(B) TIRF microscopy image of Snf7-Alexa488 dots (green) nucleated by ESCRT-II, [Snf7] = 75 nM, [Vps20] = 1 μM, [ESCRT-II] = 1 μM. Inset: zoom on 4 diffraction-limited spots (scale bar, 2 μm).(C) Histogram of the estimated number of Snf7 molecules within the dots nucleated by ESCRT-II (n = 1856).(D) HS-AFM nanodissection experiment (see text) of Snf7 spirals. 2 cycles of high AFM force were applied, between 0 s and 10 s, and between 10 s and 20 s.(E) HS-AFM time-lapse sequence showing the apparition of a new Snf7 spiral from pre-existing ones. Arrowheads show: filament protruding from a spiral (t = 8.5 s), filament curling from its tip (t = 17.0 s), and forming a small spiral (t = 37.4 s), growth of a second turn in the spiral (t = 152.2 s) and filament rearrangements (t = 164.9 s).(F) HS-AFM time-lapse sequence of an isolated Snf7 spiral. Arrowheads show: growth of the spiral at the two-turn stage (t = 67.2 s), and filament split (t = 75.7 s) leading to the three turns stage.(G) The equatorial kymograph (yellow rectangle) of this growing spiral: the innermost turn radius decreases from 22 nm to 14 nm upon formation of the third turn.(H) Dynamics of filament splitting and fusing in two Snf7 spirals (rows) observed by HS-AFM. Arrowheads show displacement of the splitting points.(I) Time plot of the outer radius of five growing Snf7 spirals followed by HS-AFM. The origin of all curves is the apparition of the first turn. The thick curve is the average of all curves. [Snf7] = 1 μM. See also Figure S4.

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 Growth of Snf7 Spirals on Supported Membranes(A) Left: TIRF microscopy kymographs of the nucleation of single Snf7 patches (green) at [Snf7] = 300 nM. Arrows indicate single ring to multiple spirals transition as postulated from the interpretation of these observations (right).(B) TIRF microscopy image of Snf7-Alexa488 dots (green) nucleated by ESCRT-II, [Snf7] = 75 nM, [Vps20] = 1 μM, [ESCRT-II] = 1 μM. Inset: zoom on 4 diffraction-limited spots (scale bar, 2 μm).(C) Histogram of the estimated number of Snf7 molecules within the dots nucleated by ESCRT-II (n = 1856).(D) HS-AFM nanodissection experiment (see text) of Snf7 spirals. 2 cycles of high AFM force were applied, between 0 s and 10 s, and between 10 s and 20 s.(E) HS-AFM time-lapse sequence showing the apparition of a new Snf7 spiral from pre-existing ones. Arrowheads show: filament protruding from a spiral (t = 8.5 s), filament curling from its tip (t = 17.0 s), and forming a small spiral (t = 37.4 s), growth of a second turn in the spiral (t = 152.2 s) and filament rearrangements (t = 164.9 s).(F) HS-AFM time-lapse sequence of an isolated Snf7 spiral. Arrowheads show: growth of the spiral at the two-turn stage (t = 67.2 s), and filament split (t = 75.7 s) leading to the three turns stage.(G) The equatorial kymograph (yellow rectangle) of this growing spiral: the innermost turn radius decreases from 22 nm to 14 nm upon formation of the third turn.(H) Dynamics of filament splitting and fusing in two Snf7 spirals (rows) observed by HS-AFM. Arrowheads show displacement of the splitting points.(I) Time plot of the outer radius of five growing Snf7 spirals followed by HS-AFM. The origin of all curves is the apparition of the first turn. The thick curve is the average of all curves. [Snf7] = 1 μM. See also Figure S4.
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fig4: Nucleation and Growth of Snf7 Spirals on Supported Membranes(A) Left: TIRF microscopy kymographs of the nucleation of single Snf7 patches (green) at [Snf7] = 300 nM. Arrows indicate single ring to multiple spirals transition as postulated from the interpretation of these observations (right).(B) TIRF microscopy image of Snf7-Alexa488 dots (green) nucleated by ESCRT-II, [Snf7] = 75 nM, [Vps20] = 1 μM, [ESCRT-II] = 1 μM. Inset: zoom on 4 diffraction-limited spots (scale bar, 2 μm).(C) Histogram of the estimated number of Snf7 molecules within the dots nucleated by ESCRT-II (n = 1856).(D) HS-AFM nanodissection experiment (see text) of Snf7 spirals. 2 cycles of high AFM force were applied, between 0 s and 10 s, and between 10 s and 20 s.(E) HS-AFM time-lapse sequence showing the apparition of a new Snf7 spiral from pre-existing ones. Arrowheads show: filament protruding from a spiral (t = 8.5 s), filament curling from its tip (t = 17.0 s), and forming a small spiral (t = 37.4 s), growth of a second turn in the spiral (t = 152.2 s) and filament rearrangements (t = 164.9 s).(F) HS-AFM time-lapse sequence of an isolated Snf7 spiral. Arrowheads show: growth of the spiral at the two-turn stage (t = 67.2 s), and filament split (t = 75.7 s) leading to the three turns stage.(G) The equatorial kymograph (yellow rectangle) of this growing spiral: the innermost turn radius decreases from 22 nm to 14 nm upon formation of the third turn.(H) Dynamics of filament splitting and fusing in two Snf7 spirals (rows) observed by HS-AFM. Arrowheads show displacement of the splitting points.(I) Time plot of the outer radius of five growing Snf7 spirals followed by HS-AFM. The origin of all curves is the apparition of the first turn. The thick curve is the average of all curves. [Snf7] = 1 μM. See also Figure S4.
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