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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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Related in: MedlinePlus

Microscope Calibration with Labeled DNA Origamis, Related to Experimental ProceduresDNA origamis are labeled with Atto-647N (GATTAquant Brightness 9R: 9 Atto-647N molecules and GATTAquant Brightness 18R: 18 Atto-647N molecules). (A) Fluorescence histograms of diffraction limited fluorescent spots (see B) of GATTA-Brightness 9R (blue, n = 985) and GATTA-Brightness 18R (orange, n = 892). (B) Typical field of view of adhered DNA origamis appearing as diffraction limited spots. (C) and (D) Average fluorescence of DNA origamis spots (9R, blue and 18R, orange), varying linearly with laser power (expressed in percentage of maximal output) in (C) and with exposure time in (D). Error bars are SD of each fluorescence distribution measured.
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figs6: Microscope Calibration with Labeled DNA Origamis, Related to Experimental ProceduresDNA origamis are labeled with Atto-647N (GATTAquant Brightness 9R: 9 Atto-647N molecules and GATTAquant Brightness 18R: 18 Atto-647N molecules). (A) Fluorescence histograms of diffraction limited fluorescent spots (see B) of GATTA-Brightness 9R (blue, n = 985) and GATTA-Brightness 18R (orange, n = 892). (B) Typical field of view of adhered DNA origamis appearing as diffraction limited spots. (C) and (D) Average fluorescence of DNA origamis spots (9R, blue and 18R, orange), varying linearly with laser power (expressed in percentage of maximal output) in (C) and with exposure time in (D). Error bars are SD of each fluorescence distribution measured.

Mentions: Imaging is performed using an inverted microscope assembled by 3i (Intelligent Imaging Innovation, Denver, USA) and Nikon (Eclipse C1, Nikon, Tokyo, Japan). For SDC imaging, a 2-μm-thick volume stack (1 μm above and below the supported membrane) is acquired then rendered to 2D by maximum intensity projection. TIRF Imaging is performed using a motorized Nikon TIRF system. The number of molecules within Snf7 oligomers is estimated by calibrating the microscope with commercially available fluorescent DNA origamis (GATTA-Brightness 9R and 18R, GATTAquant, Braunschweig, Germany) (Figure S6).


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)

Microscope Calibration with Labeled DNA Origamis, Related to Experimental ProceduresDNA origamis are labeled with Atto-647N (GATTAquant Brightness 9R: 9 Atto-647N molecules and GATTAquant Brightness 18R: 18 Atto-647N molecules). (A) Fluorescence histograms of diffraction limited fluorescent spots (see B) of GATTA-Brightness 9R (blue, n = 985) and GATTA-Brightness 18R (orange, n = 892). (B) Typical field of view of adhered DNA origamis appearing as diffraction limited spots. (C) and (D) Average fluorescence of DNA origamis spots (9R, blue and 18R, orange), varying linearly with laser power (expressed in percentage of maximal output) in (C) and with exposure time in (D). Error bars are SD of each fluorescence distribution measured.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644223&req=5

figs6: Microscope Calibration with Labeled DNA Origamis, Related to Experimental ProceduresDNA origamis are labeled with Atto-647N (GATTAquant Brightness 9R: 9 Atto-647N molecules and GATTAquant Brightness 18R: 18 Atto-647N molecules). (A) Fluorescence histograms of diffraction limited fluorescent spots (see B) of GATTA-Brightness 9R (blue, n = 985) and GATTA-Brightness 18R (orange, n = 892). (B) Typical field of view of adhered DNA origamis appearing as diffraction limited spots. (C) and (D) Average fluorescence of DNA origamis spots (9R, blue and 18R, orange), varying linearly with laser power (expressed in percentage of maximal output) in (C) and with exposure time in (D). Error bars are SD of each fluorescence distribution measured.
Mentions: Imaging is performed using an inverted microscope assembled by 3i (Intelligent Imaging Innovation, Denver, USA) and Nikon (Eclipse C1, Nikon, Tokyo, Japan). For SDC imaging, a 2-μm-thick volume stack (1 μm above and below the supported membrane) is acquired then rendered to 2D by maximum intensity projection. TIRF Imaging is performed using a motorized Nikon TIRF system. The number of molecules within Snf7 oligomers is estimated by calibrating the microscope with commercially available fluorescent DNA origamis (GATTA-Brightness 9R and 18R, GATTAquant, Braunschweig, Germany) (Figure S6).

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