A stress assembly that confers cell viability by preserving ERES components during amino-acid starvation
- Royal Netherlands Academy of Arts and Sciences, Netherlands
- University Medical Center Utrecht, Netherlands
Figures
Figure 1 with 5 supplements
Amino-acid starvation induces the formation of a novel stress assembly in Drosophila S2 cells.
(A) Immunofluorescence (IF) visualization of Delta-myc (using an anti-Delta antibody) in S2 cells in Schneider's (normal growth conditions) or incubated with Krebs Ringer Bicarbonate buffer (KRB) …
Figure 1—figure supplement 1
Sec body formation and autophagy.
(A–A′) Quantification of Atg5 punctae formation in S2 cells incubated with rapamycin (A) and incubated with KRB with or without wortmannin (A′) for indicated time points. Note that as expected, …
Figure 1—figure supplement 2
Sec body formation and single amino-acids.
Quantification of the prevention of Sec body formation by specific amino-acids upon incubation in KRB for 4 hr. This is expressed as the percentage of cells exhibiting the normal growth Sec16 …
Figure 1—figure supplement 3
Sec body formation in vivo.
Projection of four equatorial confocal planes of Sec16 and Spectrin (D) in the follicular epithelium covering an egg-chamber from an ovary dissected from a virgin fly fattened for 3 days and …
Figure 1—figure supplement 4
Sec body formation and mammalian cells.
IF visualization of Sec16 in immortalized MEFs incubated in growth medium (DMEM) or KRB plus bafilomycin for 7 hr. Note that Sec16 is remodeled into larger structures. Scale bar: 10 μm.
Figure 1—figure supplement 5
Human Sec16A is incorporated to Sec bodies in starved Drosophila S2 cells.
IF visualization of human Sec16A-V5 transfected in Drosophila S2 cells. Note that it partially localizes to ERES in fed cells but is efficiently incorporated in Sec bodies upon starvation. Scale …
Figure 2
Sec bodies are non-membrane bound structures.
(A) Immuno-electron microscopy (IEM) visualization of Sec16 (10 nm colloidal gold) in Sec bodies in ultrathin sections of S2 cells incubated in KRB for 4 hr. Arrows point to membrane in close …
Figure 3
Sec bodies are distinct from Stress Granules and P-bodies.
(A) IF visualization of endogenous FMR1, eIF4E (green), and Tral (red) in cells growing in Schneider's and incubated in KRB for 4 hr. Note that upon starvation, Stress Granules (FMR1, eIF4E) form …
Figure 4
Sec bodies form at ERES but COPII- and COPI-coated vesicle formation is not required.
(A) Stills of a GFP-Sec23 time-lapse video of a cell incubated in KRB (t = 0) for 60 min showing Sec body formation. (B–B″) IF visualization of miniSec16-V5 (B, B′) and ΔNC2-3-Sec16-V5 (B, B″) in S2 …
Figure 5 with 1 supplement
Sec23 and Sec24AB are key factors for Sec body formation.
(A–D) IF visualization of Sec16 in mock (A), Sec24AB (B), Sec23 (C), and Sec24CD (D) depleted S2 cells in Schneider's and incubated in KRB for 4 hr. Note that Sec body formation is inhibited upon …
Figure 5—figure supplement 1
The smaller structures generated in starved Sec24AB-depleted cells are not Sec bodies.
(A) Sec16 positive horseshoe shape structures observed by IF. B: Gallery of Sec16 positive IEM profiles: smaller clusters of membrane, larger ones probably corresponding to ERES mixed with Golgi …
Figure 6 with 3 supplements
Sec body proteins contain low complexity sequences that are necessary for Sec body recruitment.
(A–A′) Schematic representation of the Low Complexity Sequences (blue bars) in Sec16, Sec24AB, Sec24CD, and Sec23 (A). The red bars mark the boundaries of the Sec16 domains. Genome wide analysis of …
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Figure 6—Source data 1
Table showing the LCS content of proteins related to Stress Granules and P-bodies as well as the early secretory pathway (related to Figure 6A,A′).
- https://doi.org/10.7554/eLife.04132.016
Figure 6—figure supplement 1
LCS content of Sec24 in different species.
(A) A restricted phylogenic tree of the Sec24 sequences analyzed in (B) Note that S. pombe and S. cerevisiae Sec24 are similar to each other and to H. sapiens Sec24C and D, and D. melanogaster …
Figure 6—figure supplement 2
Secondary structure prediction of Drosophila Sec24AB using HH pred.
C/c denotes the unstructured, H/h the alpha helices, and E/e, the beta sheets. Note their absence in the 405 amino-acids of the N-terminus corresponding to LCS.
Figure 6—figure supplement 3
Sec24AB LCS is not sufficient to drive Sec body formation.
S2 cells were depleted of endogenous Sec24AB. When starved (KRB), this resulted in the formation of small structures (as in Figure 5). The transfection of Sec24AB LCS-sfGFP in these depleted cells …
Figure 7
Sec bodies have FRAP properties consistent with liquid droplets.
(A–A′) Percentage of fluorescence recovery after photobleaching (FRAP) over time of individual Sec bodies, marked by GFP-Sec23 (Video 2), and ΔNC1-Sec16-sfGFP (Video 3) in S2 cells incubated in KRB …
Figure 8 with 1 supplement
Sec bodies are functionally reversible and act as reservoir for ERES components during starvation.
(A) Stills of a GFP-Sec23 time-lapse video (Video 6) of two cells recovering in Schneider's after 4 hr in KRB (t = 0, up to 60 min). Note that Sec bodies are reversed into ERES. (B) IF localization …
Figure 8—figure supplement 1
IF localization of Sec16 in cells recovering in Schneider's for 10–30 min after 4 hr in KRB.
At 30 min, 90% of ERES are rebuilt. Note that the intensity of fluorescence of Sec16 is higher than in the cells that were maintained in Schneider's, reflecting the storage of the ERES components in …
Figure 9
Sec body formation is a pro-survival mechanism.
(A) Western blot of Sec31, Sec23, and tubulin (loading control) of lysates from GFP, Sec16, Sec23, and Sec24AB-depleted cells grown in Schneider's (S) and incubated with KRB for 4 hr (K). (B) Graph …
Videos
Video 1
Time-lapse movie of Sec body formation.
GFP-Sec23 time-lapse video of two cells incubated in KRB (t = 0) for 1 hr. One frame was taken every 3 min and the videos are displayed at 7 frame/s (related to Figure 4A).
Video 2
FRAP of one half-bleached Sec body and one fully bleached.
FRAP video of two GFP-Sec23 positive Sec bodies, one photobleached partially and one entirely, recorded every 10 ms for at least 20 s, and then every minute. The video is displayed at 7 frame/s …
Video 3
FRAP of a fully bleached Sec body.
FRAP video of one ΔNC1-Sec16-sfGFP positive Sec body entirely bleached, recorded every 10 ms for at least 20 s, and then every minute. The video is displayed at 7 frames/s (related to Figure 7B′).
Video 4
FRAP of a fully bleached Stress Granule.
FRAP video of one FMP1-sfGFP positive Stress Granule entirely bleached, recorded every 10 ms for at least 20 s, and then every minute. The video is displayed at 7 frames/s (related to Figure 7B′).
Video 5
FRAP of a partially bleached Stress Granule.
FRAP video of one FMP1-sfGFP positive Stress Granule partially bleached, recorded every 10 ms for at least 20 s, and then every minute. The video is displayed at 7 frames/s (related to Figure 7B′).
Video 6
Sec bodies are reversible.
GFP-Sec23 time-lapse video of two cells recovering in Schneider's after 4 hr in KRB (t = 0, up to 66 min). One frame was taken every 3 min and the video is displayed at 7 frame/s (related to Figure …
