Science - 31 January 2020

Leeet al.,Science 367 , eaay7108 (2020) 31 January 2020 8of10

ER tubules

RNP granule

Linescan of fluorescence intensities to

assess ER tubule localization

AB

GFP-G3BP

mCh-KDEL

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2.5 5.0

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GH

RNP granule

P-bodies

Stress granules

Fission

Events

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38

ER-

marked

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t = 0s t = 30s t = 35s t = 40s t = 45s

Fluorescence

Intensity

Pre-fission

t = 30s

Post-fission

t = 45s

ER tubules

Stress granules

GFP-Dcp2 / BFP-KDEL /

E RA-Sec61β+GB-Dcp1b t = 0s t = 6s t = 75s t = 108s t = 111s

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ER-PB contact

F P-bodies

Pre-fission Post-fission

1.1 2.2

Microns

1.1 2.2

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1.1 2.2

Microns

1.1 2.2

Microns

1.1 2.2

Microns

RNP granule Fission

Events

ddFP-

marked

P-bodies 15 15

t = 0s t = 35s t = 40s t = 45s t = 50s

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1.2 2.4

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Pre-fission

t = 40s

Post-fission

t = 50s ER tubules

P-bodies

Fluorescence

Intensity

GFP-Dcp2

mCh-KDEL

Fig. 6. ER tubules localize to the sites of PB and stress granule fission.
(A) Cartoon depicting line scan positioned perpendicular to RNP granule fission
sites to assess ER tubule localization pre- and postfission. (B) Catalog of ER
localization to PB and stress granule fission events as well as ddFP resolving of
ER contact at PB fission events. (C) Representative merged images of the ER
(red) labeled with mCh-KDEL and PBs (green) colabeled with GFP-Dcp1b
(shown), BFP-Dcp1a, and JF646-SNAP-Dcp2 in U-2 OS cells. Insets are time-
lapse images of the ER alone (top) and ER-PBs merged (bottom). Arrows
highlight ER tubules positioned at PB fission sites (Movie 4). (D) Line scan
analyses of fluorescence intensities of PBs (green) and the ER (red) pre- and
postfission. Red arrows highlight ER tubules localized to the site of PB fission.
(E) Representative images of U-2 OS cells expressing general PB (GFP-Dcp2)
and ER markers (BFP-KDEL) together with the ddFP pair from Fig. 2 to resolve

ER-PB contact (RA-Sec61band GB-Dcp1b). Insets are time-lapse merged

images of PBs (green) and ER-PB contact (red) (top); the ER (blue) and ER

contact (red) (middle); and the ER (blue), PBs (green), and ER-PBs (red)

(bottom) (Movie 5). (F) Line scan analyses of fluorescence intensities of PBs

(green) and ER-PB contact (red) for each time point. Red arrows highlight ER

tubules localized to the site of PB fission. (G) Representative merged images of

the ER (red) labeled with mCh-KDEL and stress granules (green) labeled with

GFP-G3BP in U-2 OS cells treated with 0.5 mM NaAsO 2 for 60 min followed by

40 min of washout with 200 nM ISRIB. Arrows highlight ER tubules positioned at

stress granule fission sites (Movie 6). (H) Line scan analyses of fluorescence

intensities of stress granules (green) and the ER (red) pre- and postfission. Red

arrows highlight ER tubules localized to the site of stress granule fission. In (C),

(E), and (G), scale bars are 5 and 2mm in full cell and inset images, respectively.

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