Stapled Golgi cisternae remain in place as cargo passes through the stack
- Yale University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Aggregated-CD8Lumenal as an ER staple.
(A) Domains harbored by CD8lumenal. Predicted models of trans-interacting CD8lumenal preoteins that are expected to form lumenal staples. Predicted intra-lumenal space varies from 10 to 23 nm. (B) …
Figure 1—figure supplement 1
Cytosolic staples stack the ER.
HeLa cells expressing CD8cytosolic were incubated without the disaggregating drug. Electron-micrograph showing that cytosolic staples trigger artificial stacking of ER membranes.
Figure 2 with 1 supplement
Disaggregated-CD8lumenal as an anterograde cargo.
(A) Confocal micrograph showing HeLa cells expressing CD8lumenal in the presence or in the absence of the disaggregating drug. Without drug, ER-aggregated CD8lumenal remained at the ER (ER staples), …
Figure 2—figure supplement 1
Lifetime of staples and disaggregated CD8lumenal.
HeLa cells expressing CD8lumenal were radiolabeled with 35S methionine for 30 min in the presence or the absence of the disaggregating drug (pulse). Then cells were washed and incubated at 20°C in …
Figure 3 with 1 supplement
Re-aggregated CD8lumenial as a Golgi staple, and its retention within the Golgi.
(A) General procedure: 16°C temperature block and disaggregation/re-aggregation cycles are combined to form the staples in the cis-Golgi and assess the cisternal progression model. HeLa cells …
Figure 3—figure supplement 1
Glycosylation profile of CD8lumenal.
HeLa cells expressing CD8lumenal were incubated with the disaggregating drug at 16°C for 2 hr prior to being shifted at 37°C for 2 hr in the presence (1) or absence (2) of the drug. As negative …
Figure 4
cis-Golgi retention of Golgi staples at the light microscopy level.
(A) Confocal micrograph showing HeLa cells co-expressing DsRed-Golgin97, a trans-Golgi marker, with GFP-Golgin97 (upper panel) or Grasp65-GFP, a cis-Golgi marker (lower panel). Cis- and trans-Golgi …
Figure 5 with 1 supplement
cis-Golgi retention of Golgi staples at the EM level.
HeLa cells expressing CD8lumenal were incubated at 16°C in the presence of the disaggregating drug for 2 hr, and then the drug was removed for 30 min, prior to shifting the temperature to 20°C for …
Figure 5—figure supplement 1
Staples within the Golgi.
Similar to Figure 5 Hela cells expressing CD8lumenal were incubated at 16°C in the presence of the disaggregating drug for 2 hr, and then the drug was removed for 30 min, prior to shifting the …
Figure 6 with 3 supplements
Unaltered anterograde transport within short and long-term stapled-Golgi.
HeLa cells co-expressing DsRed-CD8lumenal with VSVG-GFP were incubated overnight at 39°C in the absence of the disaggregating drug (1) prior to shifting the temperature to 16°C for 2 hr in the …
Figure 6—figure supplement 1
Golgi staples do not inhibit the rate of secretion of Collagen-I and MMP2.
Saos-2 cells expressing GFP-CD8lumenal were incubated at 20°C for 1 hr in the presence of the disaggregating drug. When required Golgi staples were formed for 2 hr (2), prior to shifting the …
Figure 6—figure supplement 2
Golgi staples do not prevent redistribution of Golgi membranes within ER mediated by BFA.
HeLa cells co-expressing ManII-DsRed and GFP-CD8lumenal were incubated at 20°C in the presence of the disaggregating drug for 1 hr. Then, when required, the drug was removed to promote staples …
Figure 6—figure supplement 3
Staples are laterally mobile and do not perturb lateral diffusion of Golgi resident enzymes.
HeLa cells expressing GFP-CD8lumenal were incubated in the absence of the disaggregating to keep the protein aggregated within the ER. FRAP was performed at 20°C (ER staples). As a negative control, …
Figure 7 with 2 supplements
Unaltered transport of soluble aggregates within stapled Golgi.
HeLa cells co-expressing GFP-CD8lumenal and DsRed-hGH, both harboring the FM-aggregation domains, were incubated at 16°C for 2 hr in the presence of the disaggregating drug (3, negative control) …
Figure 7—figure supplement 1
Co-immunoprecipitation of ER-staples and ER-soluble aggregates.
HeLa cells co-expressing GFP-CD8lumenal or GFP-hGH, with DsRed-hGH, both harboring the FM-aggregation domains, were incubated overnight in the presence of the disaggregating drug (1) or in the …
Figure 7—figure supplement 2
Segregation of staples from soluble aggregates within the Golgi at the light level.
HeLa cells co-expressing GFP-CD8lumenal and DsRed-hGH, both harboring the FM-aggregation domains, were incubated at 20°C for 2 hr in the presence of the disaggregating drug prior to shifting the …
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