Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology
- Institute of Biochemistry, ETH Zurich, Switzerland
- Institute of Pharmaceutical Sciences, ETH Zurich, Switzerland
Figures
Figure 1
Targeting of membrane proteins to the INM in vitro is dependent on GTP hydrolysis.
(A) Schematic representation of the in vitro transport assay for INM proteins (Ungricht et al., 2015). (B) Targeting of LBR(1-245)-GFP and SUN2-GFP was reconstituted in semi-permeabilized HeLa …
Figure 2
ER protein mobility is reduced in the presence of GTPγS.
(A) Model illustrating the hypothetical differences in ER organization of cells with either a highly interconnected polygonal ER (left) or a more sparsely connected network of polygons and tubules …
Figure 3
Inhibition of GTP hydrolysis affects ER morphology.
(A) Schematic of experimental set-up in B. (B) 2xRFP-tev-LBR(1-245)-GFP expressing reporter cells were semi-permeabilized and allowed to recover in HeLa cell lysate and energy at 37°C. After 30 min, …
Figure 4 with 3 supplements
Atlastin 2 depletion impairs INM targeting in vitro.
(A) Representative images of 2xRFP-tev-LBR(1-245)-GFP expressing reporter cells and ATL3 knockout (KO) reporter cells that were depleted for ATL2 by RNAi as indicated. Maximum intensity projection …
Figure 4—figure supplement 1
Targeting of LBR and SUN2 to the INM is ATL2-dependent.
(A) INM targeting of LBR(1-245)-GFP and SUN2-GFP in control cells and cells depleted of ATL2, ATL3, or co-depleted of ATL2 and ATL3 by siRNAs at the indicated time points after TEV cleavage and …
Figure 4—figure supplement 2
Validation of CRISPR/Cas9 knockout of ATL3 in HeLa cells.
Genomic sequence of the ATL3-/- isogenic clone used in this study. Sanger sequencing chromatograms show indel mutations in the ATL3 gene targeted by CRISPR. The top sequence (black) indicates the …
Figure 4—figure supplement 3
NE accumulation of the LBR-derived reporter proteins in the Atlastin depletion experiment reflects INM targeting.
Cells expressing 2xRFP-tev-LBR(1-245)-GFP were semi-permeabilized with digitonin. After TEV protease treatment, in vitro INM targeting reactions were allowed to proceed for 45 min. Then, …
Figure 5 with 1 supplement
Dominant-negative Atlastins (cytATL) cause defects in ER structure, dynamics and INM targeting.
(A) Representative images of the ER in U2OS cells overexpressing GFP-KDEL together with either RFP, cytATL2-RFP or cytATL2(R244Q)-RFP. The number of polygons within a 100 μm2 area was quantified as …
Figure 5—figure supplement 1
Addition of RabGDI does not impair INM targeting in vitro.
(A) HeLa cells were transfected with Rab7-GFP or Rab11-GFP (kind gifts of Ari Helenius). After 24 hr, cells were semi-permeabilized and incubated with the indicated concentrations of recombinant, …
Figure 6 with 2 supplements
Loss of Atlastin function causes a kinetic delay in targeting of membrane proteins to the INM.
(A) Cartoon representation of the Retention Using Selective Hooks (RUSH) system (Boncompain et al., 2012), adapted here for the analysis of INM targeting in living cells. (B) HeLa cells stably …
Figure 6—figure supplement 1
INM-RUSH reporters reach the INM after biotin-mediated release from the ER.
Cells expressing STIM1-NN-Strep and LBR(1-245)-SBP-GFP or LAP2β-SBP-GFP were incubated with biotin for 90 min or overnight and then semi-permeabilized with digitonin. To discriminate between …
Figure 6—figure supplement 2
Localization of INM proteins after Atlastin depletion.
(A) Steady state levels of INM proteins at the NE are unaffected by Atlastin depletion in most unsynchronized interphase cells. Immunofluorescence staining with emerin, LAP2β, LBR, and SUN1 …
Figure 7 with 1 supplement
Atlastin depletion affects secretory trafficking.
(A) Cartoon representation of the RUSH system to allow for synchronized secretory trafficking of membrane proteins out of the ER in live cells (Boncompain et al., 2012). (B) HeLa cells expressing …
Figure 7—figure supplement 1
The VSVG(wt)-SBP-GFP reporter protein is transported to the Golgi complex after biotin-mediated release from the ER in Atlastin-depleted cells.
HeLa cells transfected with Strep-Ii as a hook and VSVG(wt)-SBP-GFP as a reporter were either left untreated or incubated in presence of 250 μM biotin for 15 min and then fixed with 4% PFA. To stain …
Videos
Video 1
ER remodeling in cells injected with GTP.
ER dynamics in U2OS cells expressing GFP-KDEL microinjected with a solution containing 10 mM GTP and fluorescent dextran. Images were acquired with a spinning disk microscope at 1 s intervals for 1 …
Video 2
Cells injected with GTPγS show drastically reduced remodeling events in the ER.
ER dynamics in U2OS cells expressing GFP-KDEL shortly (>5 min) after microinjection with a solution containing 10 mM GTPγS and fluorescent dextran. Images were acquired with a spinning disk …
Video 3
ER undergoes extensive remodeling in control cells expressing RFP.
ER dynamics in U2OS cells co-expressing GFP-KDEL and RFP. Images were acquired with a spinning disk microscope at 1 s intervals for 1 min. The video is displayed at 4 frames/s. Image scale: 10 × 10 …
Video 4
ER dynamics in cells over-expressing dominant-negative ATL2 (cytATL2-RFP) is compromised, similar to cells injected with GTPγS (Video 2).
ER dynamics in U2OS cells co-expressing GFP-KDEL and cytATL2-RFP. Images were acquired with a spinning disk microscope at 1 s intervals for 1 min. The video is displayed at 4 frames/s. Image scale: …
Video 5
ER remodeling is unaffected in cells expressing cytATL2(R244Q)-RFP.
ER dynamics in U2OS cells co-expressing GFP-KDEL and cytATL2(R244Q)-RFP. Images were acquired with a spinning disk microscope at 1 s intervals for 1 min. The video is displayed at 4 frames/s. Image …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.28202.021
