Science - USA (2018-12-21)

Schöneberget al.,Science 362 , 1423–1428 (2018) 21 December 2018 4of6

A BD

CE

composite TD

membrane

Snf7

Vps4

pipette vesicle tube bead

2 μm

F Snf7

Vps4

membrane

-67 s

15.9 pN

45 s

22.5 pN

172 s

47.6 pN

208 s

37.0 pN

269 s

47.3 pN

422 s

0.0 pN

-70 s

37.5 pN

193 s

41.4 pN

327 s

43.9 pN

410 s

46.3 pN

640 s

53.8 pN

656 s

0.0 pN

Snf7

Vps4

membrane

composite

G

011 0

before scission after scission

fl. intensity M Snf7 Vps4 fl. intensity M Snf7 Vps4

tube remnant on bead

H

011 0

before scission after scission

fl. intensity M Snf7 Vps4 fl. intensity M Snf7 Vps4

tube remnant on bead

I

193 s

41.4 pN

167 s

40.5 pN

0 15 30

0

5

frequency

time to scission [min]

2 μM, N=24

200 nM, N=14

1

2 μM

200 nM

0

0 5 152010

time [min]

norm. cum.

time to scission

JK

Snf7 Vps4

molecules in puncta

0

200

400

600

800

composite

Fig. 3. Confocal imaging of ATP-dependent mem-
brane tube scission by ESCRTs.(AtoE) Micrograph of
the experimental setup: the GUV (center, yellow) encap-
sulating the ESCRT module and caged ATP was aspirated
by a micropipette (left). A membrane nanotube was
pulled from the GUV using a bead (right) in an optical
trap. Membrane, Snf7, and Vps4 are labeled with different
fluorophores [composite (A) of individual channels shown
in (B to E)]. Scale bar: 2mm. (FandG) Progression of two
representative membrane scission events (2 of 17). UV
illumination att= 0 s. Snf7 (green) and Vps4 (cyan)
puncta became visible at the vesicle-tube junction and migrated into thetube. Scission happened at ~410 s and ~650 s, respectively. Scale bar:
1.5mm. (HandI) Tube remnants on the bead identified scission in the middle of the tube. [Brightness adjusted compared to (F) and (G).]
(J) Quantification of tube scission. (K) Protein copy numbers in puncta.

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