Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network
- Howard Hughes Medical Institute, Harvard Medical School, United States
- Harvard Medical School, United States
Figures
Figure 1 with 1 supplement
ATL is required to maintain tubules and junctions in mammalian cells.
(A) Peripheral ER network of U2OS cells expressing GFP-calreticulin from the endogenous promoter in wild type cells. Where indicated, the cells also stably expressed mCherry-tagged wild type ATL-1 …
Figure 1—figure supplement 1
Effect of ATL overexpression on ER morphology in U2OS cells.
(A) Image of a whole U2OS cell expressing GFP-calreticulin. (B) As in (A), but a cell expressing GFP-ATL-1. (C) As in (A), but a cell expressing GFP-ATL-3. Scale bar = 10 µm. (D) Peripheral ER …
Figure 2 with 1 supplement
ATL is required to maintain an ER network in Xenopus egg extracts.
(A) An ER network was generated with a crude interphase Xenopus egg extract and stained with the lipophilic fluorescent dye DiIC18. The sample was imaged with a spinning disk confocal microscope. …
Figure 2—figure supplement 1
ER network formed with Xenopus extract fractions is disassembled by ATL inactivation.
(A) An ER network was generated with interphase light membranes. Buffer, 2 µM cytATL, 2 µM cytATL R232Q, or 2 mM GTPγS were added subsequently and the membranes were stained with octadecyl …
Figure 3
Real-time disassembly of an ER network in the presence of cytATL.
A network was formed from a crude Xenopus egg extract in the presence of the dye DiIC18 in a computer-controlled microfluidics device. CytATL-GFP (5 µM) in cytosol containing an energy regenerating …
Figure 4
Intermediates during in vitro ER network formation.
(A) DiIC18-prelabeled light membranes were mixed with buffer and an energy regenerating system. The sample was imaged immediately by confocal microscopy. Scale bar = 2 µm. (B) Xenopus egg light …
Figure 5 with 1 supplement
Localization of ATL in an in vitro generated ER network.
(A) An interphase ER network was generated with a crude Xenopus egg extract containing the dye DiIC18. Endogenous ATL was visualized by including 16 nM Alexa488-labeled, affinity-purified antibodies …
Figure 5—figure supplement 1
Localization of endogenous and inactivated ATL in a Xenopus ER network.
(A) An interphase ER network was generated with cytosol, DiIC18-prelabeled light membranes, and an energy regenerating system. Endogenous ATL was visualized by including 10.5 nM Alexa488-labeled, …
Figure 6 with 1 supplement
Effect of GTP analogs on ER network formation and maintenance.
GDP-BeF3- was added at 0.67 mM or 2 mM either before (upper panels) or after (lower panels) formation of an ER network from Xenopus egg cytosol (C), light membranes (M), and an energy regenerating …
Figure 6—figure supplement 1
ER network maintenance is less sensitive to ATL inhibition than ER network formation.
GTPγS was added at 1 mM or 2 mM either before (upper panels) or after (lower panels) formation of an ER network from Xenopus egg cytosol (C), light membranes (M), and an energy regenerating system. …
Figure 7 with 1 supplement
Interplay between ATL and the reticulons.
(A) Peripheral ER network in a U2OS cell expressing GFP-calreticulin under the endogenous promoter in wild type cells or in cells stably expressing mCherry-tagged Rtn4a. The bottom row shows three …
Figure 7—figure supplement 1
Co-overexpression of ATL mutants and Rtn4a in U2OS cells.
(A) Peripheral ER network in a U2OS cell expressing a high level of mCherry-tagged Rtn4a and GFP-ATL-1 R217Q, a dimerization defective mutant. Scale bar = 10 µm. (B) Peripheral ER network in a U2OS …
Figure 8 with 1 supplement
Lnp determines the abundance of three-way tubular junctions in mammalian cells.
(A) Views of wild type U2OS and Lnp-deleted (LnpΔ) cells expressing GFP-calreticulin from the endogenous promoter. LnpΔ cells were generated by CRISPR targeting the start codon of the LNP gene. The …
Figure 8—figure supplement 1
ER morphology in U2OS cells lacking or overexpressing Lnp.
(A) Extracts of wild type U2OS cells and a Lnp-deleted U2OS clonal cell line were analyzed by immunoblotting with Lnp and Rtn4 antibodies. Equal amounts of total protein were loaded, and GSK3β was …
Figure 9 with 1 supplement
Lnp domains important for junction localization.
(A) Schematic representation of wild type (WT) Lnp and mutants tested for proper localization in the peripheral ER. CC1, CC2, coiled-coil domains 1 and 2, respectively; TM1, TM2, trans-membrane …
Figure 9—figure supplement 1
Lnp-Lnp interaction is mediated by the Zn2+-finger domain-containing region.
(A) Schematic representation of Lnp truncation mutants tested for interaction. Hemagglutinin (HA)-tagged Lnp 99–428 was used as bait for pull-downs of mCherry-tagged truncations. (B) Immunoblots of …
Figure 10 with 1 supplement
Interplay of ATL, Lnp, and Rtn.
(A) Peripheral ER in U2OS cells stably expressing Lnp-mCherry alone or together with GFP-ATL-3 or GFP-ATL-1. Scale bar = 10 micron. (B) Peripheral ER in Lnp-lacking U2OS cells expressing …
Figure 10—figure supplement 1
ATL acts upstream of Lnp in U2OS cells.
(A) Peripheral ER in U2OS cells stably expressing Lnp-mCherry and GFP-ATL-1 K80A (GTPase defective mutant). The second and fourth rows show three time points. Stationary pixels appear white, while …
Figure 11 with 1 supplement
Effect of Lnp inactivation on an in vitro generated ER network.
(A) Schematic representation of wild type and mutant Xenopus Lnp. Phos indicates the domain phosphorylated during mitosis. (B) An ER network was generated for 20 min with crude Xenopus egg extract …
Figure 11—figure supplement 1
Cytoplasmic fragments of Lnp interfere with the function of endogenous Xenopus Lnp.
(A) Light membranes (M) were incubated with an energy regenerating mix and either buffer or 5 µM cytLnp for 15 min. The membranes were stained with octadecyl rhodamine. Scale bar = 5 µm. (B) Buffer …
Figure 12 with 1 supplement
Mitotic phosphorylation of Lnp.
(A) Xenopus egg interphase cytosol, membranes, and an energy regenerating system were incubated with buffer (Ci) or non-degradable cyclin B∆90 (Cm) for 40 min. The samples were analyzed by SDS-PAGE …
Figure 12—figure supplement 1
Mitotic phosphorylation weakens Lnp-Lnp interaction.
(A) Lnp-Lnp interaction is weakened during mitosis. Purified cytLnp and SBP-tagged cytLnp (cytLnp-SBP) were incubated with interphase cytosol in the absence or presence of cyclin B∆90. The samples …
Videos
Video 1
ER in U2OS cells expressing dominant-negative ATL-1.
U2OS cell stably expressing GFP-ATL-1 K80A (GTPase-defective mutant) with most of the peripheral ER fragmented. Images were acquired with a spinning disk confocal microscope at 0.05 sec intervals …
Video 2
ER in U2OS cells expressing dominant-negative ATL-1.
U2OS cell stably expressing GFP-ATL-1 K80A (GTPase-defective mutant) with many unbranched tubules and a fragmented peripheral ER. Images were acquired with a spinning disk confocal microscope at …
Video 3
Disassembly of an in vitro generated ER network by cytATL.
An ER network was formed in a microfluidics chamber from Xenopus crude extract containing DiIC18. Xenopus egg cytosol containing 5 µM cytATL-GFP was slowly perfused into the chamber using a …
Video 4
Intermediates of an in vitro generated ER network at early time points.
An ER network assembly reaction was prepared by mixing DiIC18-prelabeled Xenopus egg light membranes with buffer and an energy regenerating system. The sample was imaged immediately using a …
Video 5
Intermediates of an in vitro generated ER network at early time points.
As in Video 4, but the image was acquired at 0.2-sec time intervals for 5 sec. Scale bar = 5 µm. The video is shown at 5 frames per sec.
Video 6
ER morphology in U2OS cells after overexpression of Rtn4a.
U2OS cells expressing GFP-calreticulin under the endogenous promoter as well as stably expressing mCherry- Rtn4a. Left panel: calreticulin signal, right panel: mCherry-Rtn4a signal. Images were …
