Figure 4.

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.